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Objectives: To determine the impact of a rotational thromboelastometry (ROTEM)-guided transfusion protocol on the use of blood products, patient outcomes, coagulation factor concentrates, and costs. Design: A single-center retrospective cohort study. Setting: A tertiary university hospital. Patients: Adults undergoing proximal aortic surgery with deep hypothermic circulatory arrest. Intervention: ROTEM-guided transfusion protocol compared with clinically-guided transfusion. Measurements and Main Results: Two hundred seventeen patients were included; seventy-one elective and 24 emergency patients in the clinically-guided group, and 59 elective and 63 emergency patients in the ROTEM-guided transfusion protocol group. In the ROTEM-guided transfusion protocol group, a significant reduction in transfusion of red blood cells (5 [3-8] v 2 [0-4], p < 0.001), platelet concentrate (2 [2-3] v 1 [1-2], p < 0.001), and plasma (1,980 mL [1,320-3,300] v 800 mL [0-1,000], p < 0.001) was seen in elective surgery. Emergency patients received fewer red blood cells (7 [5-10] v 5 [2-10], p = 0.040), platelet concentrate (3 [2-4] v 2 [2-3], p = 0.023), and plasma (3,140 mL [1,980-3,960] v 1,000 mL [0-1,400], p < 0.001). Prothrombin complex concentrate and fibrinogen concentrate were increased significantly in elective and emergency patients. The surgical reexploration for bleeding rate was decreased in elective patients 33.8% v 5.1%. Conclusion: The implementation of a ROTEM-guided transfusion protocol might have the potential to decrease blood product transfusion and may improve patient outcomes.

Disseminated intravascular coagulation (DIC) is a syndrome triggered by infectious and noninfectious pathologies characterized by excessive generation of thrombin within the vasculature and widespread proteolytic conversion of fibrinogen. Despite diverse clinical manifestations ranging from thrombo-occlusive damage to bleeding diathesis, DIC etiology commonly involves excessive activation of blood coagulation and overlapping dysregulation of anticoagulants and fibrinolysis. Initiation of blood coagulation follows intravascular expression of tissue factor or activation of the contact pathway in response to pathogen-associated or host-derived, damage-associated molecular patterns. The process is further amplified through inflammatory and immunothrombotic mechanisms. Consumption of anticoagulants and disruption of endothelial homeostasis lower the regulatory control and disseminate microvascular thrombosis. Clinical DIC development in patients is associated with worsening morbidities and increased mortality, regardless of the underlying pathology; therefore, timely recognition of DIC is critical for reducing the pathologic burden. Due to the diversity of triggers and pathogenic mechanisms leading to DIC, diagnosis is based on algorithms that quantify hemostatic imbalance, thrombocytopenia, and fibrinogen conversion. Because current diagnosis primarily assesses overt consumptive coagulopathies, there is a critical need for better recognition of nonovert DIC and/or pre-DIC states. Therapeutic strategies for patients with DIC involve resolution of the eliciting triggers and supportive care for the hemostatic imbalance. Despite medical care, mortality in patients with DIC remains high, and new strategies, tailored to the underlying pathologic mechanisms, are needed.

BACKGROUND Milvexian (BMS-986177/JNJ-70033093) is an orally bioavailable factor XIa (FXIa) inhibitor currently in phase 2 clinical trials. OBJECTIVES To evaluate in vitro properties and in vivo characteristics of milvexian. METHODS In vitro properties of milvexian were evaluated with coagulation and enzyme assays, and in vivo profiles were characterized with rabbit models of electrolytic-induced carotid arterial thrombosis and cuticle bleeding time (BT). RESULTS Milvexian is an active-site, reversible inhibitor of human and rabbit FXIa (Ki 0.11 and 0.38 nM, respectively). Milvexian increased activated partial thromboplastin time (APTT) without changing prothrombin time and potently prolonged plasma APTT in humans and rabbits. Milvexian did not alter platelet aggregation to ADP, arachidonic acid, or collagen. Milvexian was evaluated for in vivo prevention and treatment of thrombosis. For prevention, milvexian 0.063 + 0.04, 0.25 + 0.17, and 1 + 0.67 mg/kg+mg/kg/h preserved 32 ± 6*, 54 ± 10*, and 76 ± 5%* of carotid blood flow (CBF) and reduced thrombus weight by 15 ± 10*, 45 ± 2*, and 70 ± 4%*, respectively (*p

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has caused a devastating health crisis worldwide. In this review, we have discussed that prophylactic phytochemical quercetin supplementation in the form of foods or nutraceuticals may help manage the COVID-19 pandemic. The following evidence supports our argument: first, nuclear factor erythroid-derived 2-like 2 (NRF2) agonists abrogated replication of SARS-CoV-2 in lung cells, and quercetin is a potent NRF2 agonist; second, quercetin exerts antiviral activity against several zoonotic coronaviruses, including SARS-CoV-2, mainly by inhibiting the entry of virions into host cells; third, inflammatory pathways activated by nuclear factor kappa B (NF-κB), inflammasome, and interleukin-6 (IL-6) signals elicit cytokine release syndrome that promotes acute respiratory distress syndrome in patients with COVID-19, and quercetin inhibits these pro-inflammatory signals; fourth, patients with COVID-19 develop thrombosis, and quercetin mitigates coagulation abnormalities by inhibiting plasma protein disulfide isomerase. This review provides a strong rationale for testing quercetin for the management of COVID-19., Graphical abstract Image 1

Hemophilia is a hemorrhagic disorder with a sex-linked inherited pattern, characterized by an inability to amplify coagulation due to a deficiency in coagulation factor VIII (hemophilia A or classic) or factor IX (hemophilia B). Sequencing of the genes involved in hemophilia has provided a description and record of the main mutations, as well as a correlation with the various degrees of severity. Hemorrhagic manifestations are related to levels of circulating factor, mainly affecting the musculoskeletal system and specifically the large joints (knees, ankles, and elbows). This document is a review and consensus of the main genetic aspects of hemophilia, from the inheritance pattern to the concept of women carriers, physiopathology and classification of the disorder, the basic and confirmation studies when hemophilia is suspected, the various treatment regimens based on infusion of the deficient coagulation factor as well as innovative factor-free therapies and recommendations for the management of complications associated with treatment (development of inhibitors and/or transfusion-transmitted infections), or secondary to articular hemorrhagic events (hemophilic arthropathy). Finally, relevant reviews of clinical and treatment aspects of hemorrhagic pathology characterized by acquired deficiency of FVIII secondary to neutralized antibodies named acquired hemophilia.

Thrombus formation is dependent on the interaction of platelets, leukocytes and endothelial cells as well as proteins of the coagulation cascade. This interaction is tightly controlled by phospho-regulated pathways involving protein kinase CK2. A growing number of studies have demonstrated an important role of this kinase in the regulation of primary and secondary hemostasis. Inhibition of CK2 downregulates the expression of important adhesion molecules on platelets and endothelial cells, such as glycoprotein (GP)IIb/IIIa, P-selectin, von Willebrand factor and vascular cell adhesion molecule. Moreover, the reduced CK2-dependent phosphorylation of different coagulation factors prevents the conversion of fibrinogen to fibrin. Targeting these mechanisms may open the door for the development of novel anti-thrombotic therapies.

Aim: Feedback activation of factor XI (FXI) by thrombin is believed to play a critical role in the amplification phase of thrombin generation and to contribute to thrombosis development and hemostasis. However, the activation of FXI by thrombin has been shown in vitro to require a cofactor. In this study, the role of thrombin in activated FXI (FXIa) formation in vivo is investigated. Methods: The study population comprised probands in whom coagulation activation was triggered by low-dose (15 µg/kg) recombinant activated factor VII (rFVIIa, n =89), of whom 34 with (VTE+) and 45 without a history of venous thromboembolism (VTE−), and patients undergoing major orthopedic surgeries ( n =45). FXIa was quantified via an enzyme capture assay using a monoclonal FXI-specific antibody. Thrombin formation was monitored using an oligonucleotide-based enzyme capture assay and the thrombin activation markers prothrombin fragment 1＋2 (F1＋2) and thrombin–antithrombin complex (TAT). Results: In the rFVIIa cohort, FXIa and thrombin remained below their lower limit of quantification of 3.48 and 1.06 pmol/L, respectively. By contrast, during the surgeries, median FXIa levels increased from 3.69 pmol/L pre-operatively to 9.41 pmol/L mid-operatively ( P =4·10 −4 ) and remained significantly elevated 24 h thereafter, with 9.38 pmol/L ( P =0.001). Peak levels of F1+2 were comparable in the VTE+, VTE−, and surgery cohort (235, 268, and 253 pmol/L), whereas peak TAT levels were higher in the surgery cohort (53.1, 33.9, and 147.6 pmol/L). Conclusions: Under in vivo conditions, the activation of FXI requires specific local features that are present at the wounded site including potential cofactors of thrombin.

The haemostatic system acts in concert with inflammation, so that after inflammatory response various mediators activate the haemostatic system through endothelial dysfunction, platelet activation and coagulation promoting thrombosis, which is termed thromboinflammation. In this process, the inflammasome acquires special relevance; its stimulation promotes innate and adaptive immune responses. Inflammasome activation plays an important physiopathological role in several disorders with inflammatory and thrombotic phenomena. The role of thromboinflammation has become relevant in the COVID-19 pandemic, in which a cytokine storm has been described as one of the responsible mechanisms.

Pulmonary thromboembolism (PTE) is one of the leading causes of maternal mortality. We previously reported that possible contamination of amniotic fluid (AF) into maternal circulation accelerated thrombin production and activated platelet function in maternal blood through the extrinsic pathway, which may be associated with the high incidence of PTE in early puerperium. However, it remains unclear whether the maternal anticoagulation system, e.g., the activated protein C (APC) pathway, contributes to the hypercoagulable condition induced by AF. Our previous study using an endogenous thrombin potential (ETP)-based assay revealed that sensitivity to APC was reduced during the postpartum first day, i.e., immediately after delivery, when parturients were supposed to be exposed to AF. Our aim is to investigate the susceptibility of maternal plasma to APC when mixed with AF. We collected plasma from 51 pregnant females and mixed with AF as well as APC. APC-sensitivity ratio (APC-sr) was calculated using the ETP-based assay. Addition of AF to maternal plasma showed a significant increase of ETP in the presence of APC. APC-sr was significantly increased, indicating decreased sensitivity to APC, after AF mixture to maternal plasma. The present APC-sr difference with AF contamination was smaller than that we reported previously in venous thromboembolism cases. The inhibitory effects of AF on the APC anticoagulation pathway may contribute, at least partly, to further promotion of thrombin production induced by AF. Combined with other classical thrombophilic risk factors, the present findings support possible involvements of AF exposure in the high incidence of PTE in early puerperium.

Rivaroxaban is an oral anticoagulant that inhibits thrombin and blocks coagulation cascade through directly inactivating factors Xa. Despite rivaroxaban is widely used for prevention and treatment of venous thrombosis, and its common adverse reactions have been reported, including abnormal coagulation, mucosal hemorrhage, hematuria, and intracranial hemorrhage. To explore potential drivers of individual differences in adverse reactions induced by rivaroxaban, we performed whole-exome sequencing and found that AKR7A3 rs1738023/rs1738025 and ABCA6 rs7212506 are susceptible sites for rivaroxaban-related bleeding in aged patients treated with rivaroxaban. Gene functional annotation and signaling pathway enrichment indicated that homozygous mutations in AKR7A3 and ABCA6 might alter normal rivaroxaban transport and metabolism, and lead to continuous accumulation of activated drugs and toxic substances in vivo. Our results suggested that interindividual differences in bleeding events induced by rivaroxaban may be potentially driven by genetic alterations related to abnormal metabolism and transport of rivaroxaban.

Quantra® Hemostasis Analyzer is a Point of the care device that uses ultrasound technology to assess clot formation. In this study, we establish how Quantra® system performs compared to conventional coagulation tests at low levels of fibrinogen in the blood obtained from pregnant women. 24 mL blood was obtained from each healthy parturient. Blood was analyzed for Quantra® variables (Q): Clot time (CT), Clot stiffness (CS), platelet contribution to CS (PCS), fibrinogen contribution to CS (FCS), and conventional coagulation (CL) tests: PT, aPTT, INR, Factor VIII and fibrinogen. 6 ml blood were centrifuged to obtain pregnant plasma. 30 mL of saline was added to 10 mL of blood to simulate crystalloid resuscitation (DB) and was evaluated for Q and CL. Fractions of pregnant plasma, or nonpregnant plasma (Blood Bank) was added to DB to obtain 15% and 30% clotting factor enriched samples. 4 ml of DB was added to 4 ml of original blood (1:1) to obtain the final sample (resus). Each of the samples were analyzed for Q and CL parameters. Regression analysis and Receiving Characteristics Curves were used to study the relationship between Quantra variables and CL tests. There were remarkably high linear correlations between Fibrinogen and CS (R = 0.93, P

Haemophilia A and B are congenital bleeding disorders characterized by missing or defective factor VIII or factor IX, respectively. Factor replacement therapy has been the gold standard for prophylaxis and treatment of bleeding complications. However, the inconvenience of regular intravenous administration, along with progression of arthropathy and development of inhibitors has driven the need for alternative treatment options, such as extended half-life products, non-factor coagulation products, such as subcutaneous emicizumab, blocking natural anticoagulants (rebalancing haemostatic agents) and gene therapy, which have been useful to control bleeding or are currently under late-phase clinical investigation.

Thrombin is the pivotal enzyme in the biochemistry of secondary hemostasis crucial to maintaining homeostasis of hemostasis. In contrast to routine coagulation tests (PT or aPTT) or procoagulant or anticoagulant factor assays (e.g. fibrinogen, factor VIII, antithrombin or protein C), the thrombin generation assay (TGA), also named thrombin generation test (TGT) is a so-called "global assay" that provides a picture of the hemostasis balance though a continuous and simultaneous measurement of thrombin formation and inhibition. First described in the early 1950s, as a manual assay, efforts have been made in order to standardize and automate the assay to offer researchers, clinical laboratories and the pharmaceutical industry a versatile tool covering a wide range of clinical and non-clinical applications. This review describes technical options offered to properly run TGA, including a review of preanalytical and analytical items, performance, interpretation, and applications in physiology research and pharmacy.

Apixaban is used to treat venous thromboembolism (VTE) at 10 mg twice daily (BID) for 7 days, followed by 5 mg BID without dose adjustment, and non-valvular atrial fibrillation (NVAF) at 5 mg BID or 2.5 mg BID with dose adjustment criteria (DAC) including age, body weight, and renal function. The anti-factor Xa activity (AXA), prothrombin time (PT), and activated partial thromboplastin time (APTT) in patients with VTE receiving 10 mg BID of apixaban remains unclear. Twenty-six patients (70.8±15.4 years, 10 males) with VTE receiving 10 mg BID of apixaban were enrolled. The patients were divided into two groups based on whether they met the DAC of NVAF: DAC group (n=8) and non-DAC group (n=18). Trough and peak AXA values, PT, and APTT were measured at 10 mg BID dosage and then at 5 mg BID dosage. Coagulation markers in recipients of 10 mg BID therapy were significantly higher than those of 5 mg BID recipients. A significant and strong positive correlation was observed between AXA and PT at trough and peak times. The AXA values and PT in the DAC group were significantly higher than those in the non-DAC group. No significant inter-group differences were seen in APTT. This study provides the first report of AXA distribution in VTE patients receiving 10 mg BID of apixaban. Our findings indicate that coagulation markers may differ in patients with VTE-prescribed higher doses of apixaban and a DAC may be warranted in such patients.

Purpose The study objective was to explore whether prophylaxis with vitamin K1 improves abnormal coagulation function–associated cefoperazone-sulbactam in patients treated in the long term with low-dose aspirin. Methods This retrospective, observational study assessed patients treated with long-term low-dose aspirin in a naval military hospital in China from 2004 to 2018, including all patients treated concurrently with cefoperazone-sulbactam with or without vitamin K1. Differences in the coagulation index were analyzed statistically before and after receipt of cefoperazone-sulbactam. Findings The cohort included 227 patients. After cefoperazone-sulbactam treatment, the mean (SD) prothrombin time (PT) was 14.07 (3.07) seconds, activated partial thromboplastin time (aPTT) was 35.15 (4.78) seconds, and international normalized ratio (INR) was 1.49 (0.49) in the cefoperazone-sulbactam group, which was significantly higher than the PT of 11.55 (1.29), aPTT of 31.37 (2.20), and INR of 1.12 (0.35) before cefoperazone-sulbactam treatment. No significant difference was in the cefoperazone-sulbactam plus vitamin K1 group. In addition, no significant difference was found in the thrombin time or fibrinogen level between before and after cefoperazone-sulbactam treatment in both groups. The mean (SD) platelet counts of the 2 groups were 197.34 (71.82) × 109/L and 187.75 (72.66) × 1 09/L after cefoperazone-sulbactam treatment, respectively, which was significantly lower than 231.77 (77.05) × 109/L and 232.08 (84.48) × 109/L before cefoperazone-sulbactam treatment. There were greater proportions of coagulation disorders (prolongation of PT, aPTT, INR, and bleeding) after cefoperazone-sulbactam treatment in the cefoperazone-sulbactam group compared with that in the cefoperazone-sulbactam plus vitamin K1 group. Implications Results indicate that, after adding cefoperazone-sulbactam to the regimens of patients receiving long-term low-dose aspirin, therapy contributed to remarkable increase in abnormal coagulation function and coagulation disorders. Prophylaxis with vitamin K1 decreased the risk of these abnormalities in blood coagulation parameters associated with cefoperazone-sulbactam in patients taking long-term aspirin.

Thrombin generation assay (TGA) is a useful tool to evaluate the initiation, propagation and inhibition of coagulation. TGA is a global test that is used to assess hemorrhagic risk in hemophilia patients, but it can also be used to study hypercoagulable states. The interest of TGA is to screen for cardiovascular risk, which is regularly associated with autoimmune disease (AID) such as antiphospholipid syndrome. Indeed, TGA has been used to evaluate hypercoagulability in patients with antiphospholipid syndrome treated with rivaroxaban versus warfarin. In other AIDs without thrombotic events, TGA measurement is elevated, mainly in rheumatoid arthritis (RA), systemic lupus erythematosus and Behcet's disease. These findings in RA are correlated with the inflammatory activity of the disease. In systemic lupus erythematosus and Behcet's disease, TGA appears to reflect disease activity. In conclusion, TGA remains relatively under used in the clinical evaluation of AID, but it could play a greater role in the evaluation of certain potentially thrombogenic treatments in AID. Finally, TGA helps measuring AID activity, due to the clearlink between coagulation and inflammation, despite some limitations of interpretation mainly due to a lack of standardization.

Determining patient's coagulation profile, i.e. detecting a bleeding tendency or the opposite, a thrombotic risk, is crucial for clinicians in many situations. Routine coagulation assays and even more specialized tests may not allow a relevant characterization of the hemostatic balance. In contrast, thrombin generation assay (TGA) is a global assay allowing the dynamic continuous and simultaneous recording of the combined effects of both thrombin generation and thrombin inactivation. TGA thus reflects the result of procoagulant and anticoagulant activities in blood and plasma. Because of this unique feature, TGA has been widely used in a wide array of settings from both research, clinical and pharmaceutical perspectives. This includes diagnosis, prognosis, prophylaxis, and treatment of inherited and acquired bleeding and thrombotic disorders. In addition, TGA has been shown to provide relevant information for the diagnosis of coagulopathies induced by infectious diseases, comprising also disturbance of the coagulation system in COVID-19, or for the assessment of early recurrence in breast cancer. This review article aims to document most clinical applications of TGA.

Various topical hemostatic agents can help induce coagulation at the liver cut surface. However, hemostasis is usually ineffective in controlling bleeding. We present a case of rescue fibrin glue-infiltrating hemostasis combined with hepatorrhaphy to manage intractable postoperative bleeding from the liver cut surface. The case was a 56-year-old male patient with hepatocellular carcinoma in hepatitis B virus-associated cirrhotic liver. The patient was administered warfarin because of graft replacement of the ascending aorta and hemi-arch one year earlier. After warfarin was discontinued, segment VII partial hepatectomy was performed according to standard procedures. However, considerable bleeding occurred during and after hepatectomy. Bleeding from the liver cut surface was controlled over one hour using surface coagulation and topical application of four kinds of hemostatic agents. However, active abdominal bleeding led to reoperation soon after the hepatectomy. During the reoperation, we identified diffuse oozing from the edge of the liver cut surface which was difficult to control. Thus, we performed direct parenchymal injection of fibrin glue at the bleeding points using 12 fibrin glue kits which induced complete hemostasis. Because the patient would undergo anticoagulation again soon after the operation, we also performed hepatorrhaphy. The patient recovered uneventfully after the reoperation. He has been doing well for six months without complications. In conclusion, fibrin glue-infiltrating hemostasis effectively controlled intractable bleeding from the hepatic cut surface in our case. Thus, it can be considered as an optional method for rescue hemostasis.

SARS-CoV-2 causes thrombotic microangiopathy (TMA) through the activation of an alternative and lectin complement pathway. TMA is one of the main reasons for acute kidney injury development in patients with COVID-19. In this study, we present 3 TMA cases with severe kidney injury triggered by SARS-CoV-2. In the absence of other TMA causes, we diagnosed the atypical hemolytic uremic syndrome, triggered by SARS-CoV-2 due to abnormal complement activation. Because of both coagulation factors activation, and the high level of D-dimer in patients with COVID-19, it is crucial to differentiate disseminated intravascular coagulation from TMA. The use of anticomplement therapies such as eculizumab should be considered in refractory cases of progressive COVID-19. Controlled clinical trials are required before a definitive statement can be made.

Leeches are amazing animals that can be classified as conditionally poisonous animals since the salivary cocktail they produce is injected directly into the victim, and its components have strictly defined biological purposes, such as preventing blood clot formation. Thrombolytic drugs are mainly aimed at treating newly formed blood clots. Aged clots are stabilized by a large number of isopeptide bonds that prevent the action of thrombolytics. These bonds are destroyed by destabilase, an enzyme of the leech’s salivary glands. Here, we conducted a pilot study to evaluate the feasibility and effectiveness of the use of destabilase in relation to blood clots formed during real pathological processes. We evaluated the isopeptidase activity of destabilase during the formation of a stabilized fibrin clot. We showed that destabilase does not affect the internal and external coagulation cascades. We calculated the dose–response curve and tested the ability of destabilase to destroy isopeptide bonds in natural blood clots. The effect of aged and fresh clots dissolving ability after treatment with destabilase coincided with the morphological characteristics of clots during surgery. Thus, recombinant destabilase can be considered as a potential drug for the treatment of aged clots, which are difficult to treat with known thrombolytics.

The antiphospholipid syndrome is characterized by antibodies directed against phospholipid-binding proteins and phospholipids attached to cell membrane receptors, mitochondria, oxidized lipoproteins, and activated complement components. When antibodies bind to these complex antigens, cells are activated and the coagulation and complement cascades are triggered, culminating in thrombotic events and pregnancy morbidity that further define the syndrome. The phospholipid-binding proteins most often involved are annexins II and V, β2-glycoprotein I, prothrombin, and cardiolipin. A distinguishing feature of the antiphospholipid syndrome is the “lupus anticoagulant.” This is not a single entity but rather a family of antibodies directed against complex antigens consisting of β2-glycoprotein I and/or prothrombin bound to an anionic phospholipid. Although these antibodies prolong in vitro clotting times by competing with clotting factors for phospholipid binding sites, they are not associated with clinical bleeding. Rather, they are thrombogenic because they augment thrombin production in vivo by concentrating prothrombin on phospholipid surfaces. Other antiphospholipid antibodies decrease the clot-inhibitory properties of the endothelium and enhance platelet adherence and aggregation. Some are atherogenic because they increase lipid peroxidation by reducing paraoxonase activity, and others impair fetal nutrition by diminishing placental antithrombotic and fibrinolytic activity. This plethora of destructive autoantibodies is currently managed with immunomodulatory agents, but new approaches to treatment might include vaccines against specific autoantigens, blocking the antibodies generated by exposure to cytoplasmic DNA, and selective targeting of aberrant B-cells to reduce or eliminate autoantibody production.

Background Platelet collection and processing methods, as well as donor attributes, can influence platelet function and quality during ex vivo storage. In this study, activation and procoagulant responses in platelets collected from donors experiencing a citrate reaction (CR) were investigated. Study design and methods Apheresis platelet components (n = 54) were stored in 100% autologous plasma and tested on days 1 and 5 post-collection. Platelet components were categorized into two groups according to whether the donor had experienced a CR during donation (n = 10; non-CR group, n = 44). Platelet aggregation was initiated with collagen and thrombin. Platelet phenotype was characterized by flow cytometry. Fibrinogen binding was assessed following collagen + thrombin stimulation (COATed platelets), and procoagulant activity was assessed using a procoagulant phospholipid assay (PPL). Platelet microparticle (PMP) subsets were enumerated by flow cytometry. Results Basal von Willebrand factor (VWF) binding was higher in the CR donations when compared with the non-CR group. Collagen aggregation was significantly higher in platelets from CR donations, in contrast to aggregation induced by thrombin. The proportion of phosphatidylserine (PS) positive PMP and PPL clotting time were higher in the CR group, in contrast to the number of basal PS+ platelets and COATed platelets following stimulation. Conclusion Platelets donated by donors who experienced a CR during donation had higher platelet activation response and possibly a more procoagulant PMP phenotype, suggesting that this donor reaction might lead to increased platelet activation.

Background Critically ill horses, such as horses with gastrointestinal (GI) disease, often suffer from hemostatic aberrations. Global hemostatic tests examining the initiation of coagulation, clot strength and fibrinolysis, such as the Calibrated Automated Thrombogram (CAT) and plasma-thromboelastography (TEG) have not been evaluated in horses. This study aimed to evaluate CAT and apply plasma-TEG in horses. Test performance of CAT was evaluated on equine platelet poor plasma with intra- and inter-assay variability (CV) and a heparin dilution curve. To examine clinical performance of both tests, group comparisons were assessed comparing healthy horses, horses with mild and severe GI disease with both CAT and plasma-TEG. Results For CAT, intra- and inter-assay CVs were established for lag-time (1.7, 4.7%), endogenous thrombin potential (1.6, 4.6%), peak (2.6, 3.9%) and time to peak (ttPeak) (1.9, 3.4%). Increasing heparin concentrations led to the expected decrease in thrombin generation. In the group comparison analysis, CAT showed significant higher peak (p = 0.04) and ttPeak (p = 0.008) in the severe GI disease group compared to horses with mild GI disease and healthy horses, respectively. Plasma-TEG showed an increased angle (p = 0.032), maximum amplitude (p = 0.017) and shear elastic force (G) (p = 0.017) in the severe GI disease group compared to healthy horses. Conclusions CAT performed well in horses. Both CAT and plasma-TEG identified hemostatic aberrations in horses with severe GI disease compared to healthy horses. Further studies including more horses, are needed to fully appreciate the use of CAT and plasma-TEG in this species.

Rosuvastatin therapy might have an effect on the inflammatory and coagulation biomarkers. However, the evidence about the effect of rosuvastatin therapy on the high-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), and D-dimer levels among people living with human immunodeficiency virus (PLHIV) is still unclear. Therefore, this study investigated the relational effect of rosuvastatin therapy on serum/plasma hsCRP, IL-6 and D-dimer levels in PLHIV. The literature search was done from Embase, PubMed, and Web of Science databases. The review and meta-analysis included studies written in English language up to January 4, 2020. Random effects model was used to evaluate the pooled standard mean difference with 95% confidence interval. A meta-analysis was performed using nine articles with 392 PLHIV. The result revealed that the plasma/serum levels of IL-6 were significantly reduced after the intervention. However, hsCRP and D-dimer levels showed no significant difference (

INTRODUCTION Chronic liver disease (CLD) is characterized by changes in haemostasis, embracing both hypo- and hypercoagulability. Global hemostatic tests such as thrombin generation assays evaluate the hemostatic balance, to better assess bleeding and thrombotic risks. In addition, procoagulant state in patients with CLD has been demonstrated using modified thrombin generation assays with thrombomodulin, a cofactor for protein C activation. In this study, we prospectively determined thrombin generation and thrombomodulin resistance in patients with CLD staged with liver stiffness measurement (LSM), using both the fully automated analyzer ST Genesia® Thrombin Generation System (STG) and the calibrated automated thrombogram assay (CAT). MATERIALS AND METHODS Demographic, clinical and laboratory characteristics, and blood samples were collected from 65 patients with CLD. Liver stiffness was measured by transient elastography, and thrombin generation and thrombomodulin resistance, by STG and CAT. RESULTS Patients were separated based on LSM of

Global coagulation potential was assessed in 59 patients with acquired hemophilia A (PwAHA) by clot waveform analysis (CWA) and/or thrombin and plasmin generation assay. Relationships between factor VIII activity (FVIII:C) and the parameters from CWA and T/P-GA in patients with congenital HA were compared by grading coagulation potential related to FVIII:C: T1 (FVIII:C < 1 IU/dL), T2 (1 ≤ , ≤ 5 IU/dL), T3 (5 < , 12 ≤ IU/dL), and T4 (12 < , ≤ 50 IU/dL). The median FVIII:C and inhibitor titers in PwAHA on admission were 3.3 IU/dL and 63.0 BU/mL, respectively, but global coagulation parameters corresponded to T1 or less. Median FVIII:C levels during follow-up in PwAHA were 1.7-9.6-6.7-40.0-21.7 IU/dL on days 0-14-28-56-93, respectively. CWA-based data corresponded to less than T2 until day 28, but more closely reflected FVIII:C after day 56. Peak thrombin was severely low (near T1) until day 28 and improved modestly after day 56 but remained less than T2. Peak plasmin was lower than T1 until day 56, and returned to T4 on day 93. In conclusion, global coagulation function in PwAHA was impaired to a greater extent than could be anticipated from assays of FVIII:C, until approximately 1 month after immunosuppression and treatment with FVIII-bypassing agents.

Background Few studies detail the evolution of COVID-19 associated coagulopathy. We performed serial thromboelastography (TEG) and laboratory coagulation studies in 40 critically-ill, mechanically ventilated COVID-19 patients over a 14-day period and analysed differences between 30-day survivors and non-survivors. Methods Single-center prospective, observational study including 40 patients with severe COVID-19 pneumonia admitted to the intensive care unit (ICU) for mechanical ventilation. TEG analysis was performed on days 1, 7 and 14 of ICU admission and laboratory coagulation studies were performed on days 1 and 14. Coagulation variables were evaluated for change over the 14-day observation period. Differences between survivors and non-survivors at 30-days were analysed and compared. Results On admission, TEG maximum amplitude (MA) with heparinase correction was above the upper limit of the reference range in 32 (80%) patients while 33 (82.5%) presented with absent clot lysis at 30 min. The functional fibrinogen MA was also elevated above the upper limit of the reference range in 37 (92.5%) patients. All patients had elevated D-dimer and fibrinogen levels, mildly prolonged prothrombin times (PT), normal platelet counts and normal activated partial thromboplastin times (aPTT). The heparinase MA decreased significantly with time and normalised after 14 days (p = p = 0.113). No significant differences in TEG analysis were noted between 30-day survivors and non-survivors at all time points. No patients developed disseminated intravascular coagulopathy (DIC) after 14-days, however thrombosis and bleeding were each reported in 3 (7.5%) patients. Conclusion Critically-ill patients with COVID-19 present in a hypercoagulable state characterised by an increased clot strength. This state normalises after 14 days despite a persistently increased fibrin contribution to clot strength. We were unable to demonstrate any significant differences in TEG parameters between 30-day survivors and non-survivors at all time points.

Haemonchus contortus, a blood-feeding nematode, inhibits blood coagulation at the site of infection to facilitate blood-sucking and digesting for successful parasitism. However, the mechanism underlying anti-coagulation at the host-parasite interface is largely unknown. In the current study, Hc-spi-i8, which has two greatly different transcripts named Hc-spi-i8a and Hc-spi-i8b, respectively, was described. Hc-SPI-I8A was a serine protease inhibitor containing a trypsin inhibitor-like cysteine rich (TIL) domain, while Hc-SPI-I8B was not. Hc-SPI-I8A/B were primarily expressed in the hypodermis, intestines and gonads in the parasitic stages of H. contortus. Hc-SPI-I8A interacted with Ovis aries TSP1-containing protein (OaTSP1CP), which was determined by yeast two-hybrid, co-immunoprecipitation (Co-IP), pull down and co-localization experiments. The blood clotting time contributed by the TIL domain was prolonged by Hc-SPI-I8A. Hc-SPI-I8A is most likely interfering in the extrinsic coagulation cascade by interacting with OaTSP1CP through its TIL domain and intrinsic coagulation cascade by an unknown mechanism. These findings depict a crucial point in the host-parasite interaction during H. contortus colonization, which should contribute to drug discovery and vaccine development in fighting against this important parasite worldwide.

Objective: Pancreatic cancer activates coagulation and increases risk of venous thromboembolism (VTE). We aimed at characterizing the association of hemostatic biomarkers and VTE with mortality and chemotherapy response. Approach and Results: Pancreatic cancer patients (N=145) were included in a prospective, observational cohort study (CATS [Vienna Cancer and Thrombosis Study]). Hemostatic biomarkers (D-dimer, extracellular vesicle–tissue factor activity, prothrombin fragment 1+2, fibrinogen, factor VIII, PAI-1 [plasminogen activator inhibitor 1], sP-selectin [soluble P-selectin], thrombin generation assay) were measured at inclusion. The impact of VTE on overall survival/progression-free survival (OS/PFS) was evaluated by multistate modeling. The association of biomarkers with OS was analyzed by Cox-regression and with PFS and disease control rate in patients initiating palliative chemotherapy (n=95) by Cox-regression and logistic regression. Multivariable analysis included stage, grade, sex, age, performance status, VTE (time-dependent), vascular infiltration/compression, and tumor marker levels (carbohydrate-antigen 19-9, carcinoembryonic antigen). VTE occurrence was associated with shorter OS (transition hazard ratio, 3.40 [95% CI, 2.05–5.64]) and shorter PFS (transition hazard ratio, 2.10 [1.16–3.79]). Median post-VTE OS/PFS in months was 5.5 [2.2–6.5] and 3.0 [1.5–3.9], compared with 13.4 [9.7–16.6] and 7.5 [5.9–9.8] in patients without VTE (both P Conclusions: VTE diagnosis is associated with shorter OS and PFS. Higher baseline levels of D-dimer, extracellular vesicle–tissue factor activity, PAI-1, and sP-selectin were independently prognostic for increased mortality, and D-dimer predicted response to palliative chemotherapy. Graphic Abstract: A graphic abstract is available for this article.

Background: Hydroxyethyl starch solutions which have been introduced some three decades ago, have been extensively used for treatment of intraoperative hypovolaemia. Aims and Objectives: The present prospective study was conducted to study changes in post-operative renal function and coagulation profile in patients undergoing orthopedic hip surgery under spinal anesthesia after coloading with 6% HES 130/0.4. Materials and Methods: Seventy patients, aged between 50 and 80 years, of either sex, belonging to ASA physical Status I and II, scheduled for orthopedic hip surgery under spinal anesthesia were included in the study. As per the group allocation, coloading was done with 7.5 mlkg-1 of either Group RL or 6% 130/0.4 Group HES during the 1st h of surgery. Venous samples were withdrawn before the day of surgery, on the day of surgery, and on the 3rd day of surgery. Results: No statistically significant difference between renal and coagulation profile was found among groups. Conclusion: Thus, we recommend using tetrastarch (6% HES 130/0.4) for intraoperative hypovolemia in elderly patients with normal renal function and coagulation profile undergoing orthopedic hip surgery.

Patients admitted to the intensive care unit (ICU) with coronavirus disease 2019 (COVID-19), the infectious pathology caused by severe acute respiratory syndrome coronavirus 2, have a high risk of thrombosis, though the precise mechanisms behind this remain unclarified. A systematic literature search in PubMed and EMBASE identified 18 prospective studies applying dynamic coagulation assays in ICU COVID-19 patients. Overall, these studies revealed normal or slightly reduced primary hemostasis, prolonged clot initiation, but increased clot firmness. Thrombin generation assay parameters generally were equivalent to the control groups or within reference range. Fibrinolysis assays showed increased clot resistance. Only six studies related their findings to clinical outcome. We also prospectively included 51 COVID-19 patients admitted to the ICU. Blood samples were examined on day 1, 3–4, and 7–8 with platelet function tests, rotational thromboelastometry (ROTEM), in vivo and ex vivo thrombin generation, and clot lysis assay. Data on thrombosis, bleeding, and mortality were recorded during 30 days. Primary hemostasis was comparable to healthy controls, but COVID-19 patients had longer ROTEM-clotting times and higher maximum clot firmness than healthy controls. Ex vivo thrombin generation was similar to that of healthy controls while in vivo thrombin generation markers, thrombin–antithrombin (TAT) complex, and prothrombin fragment 1 + 2 (F1 + 2) were higher in ICU COVID-19 patients than in healthy controls. Impaired fibrinolysis was present at all time points. TAT complex and F1 + 2 levels were significantly higher in patients developing thrombosis (n = 16) than in those without. In conclusion, only few previous studies employed dynamic hemostasis assays in COVID-19 ICU-patients and failed to reveal a clear association with development of thrombosis. In ICU COVID-19 patients, we confirmed normal platelet aggregation, while in vivo thrombin generation was increased and fibrinolysis decreased. Thrombosis may be driven by increased thrombin formation in vivo.

INTRODUCTION: Coronavirus 2 (CoV-2) infection or coronavirus disease 2019 (COVID-19) is frequently associated with microvascular thrombosis.The microthrombosis in COVID-19 is the result of the interplay between inflammation and endotheliopathy. Elevated interleukin-6 (IL-6) characterizes COVID-19 inflammation resulting in endotheliopathy and coagulopathy marked by elevated D-dimer (DD). Aim of this study is to identify and to describe the coagulation changes in 100 moderate COVID-19 patients having lung involvement and to determine the association of coagulopathy with the severity and prognosis. METHODS: Inflammation, endothelial and coagulation molecules were measured in moderate and mild disease. RESULTS: IL-6 and tumor necrosis factor-α (TNF-α) and tissue factor (TF), von Willebrand factor (VWF), and tissue factor pathway inhibitor (TFPI) significantly increased in moderate disease as well as D-dimer, thrombin antithrombin complex (TAT), Fibrinogen (Fib), platelet factor-4 (PF4), s-thromboglobulin (s-TG), P-selectin, and platelet adhesion. Shortened clotting time (CT) and clot formation time (CFT), high maximum clot firmness (MCF) and low LY at 30 min were present in 100% of moderate COVID-19 patients compared with mild COVID-19 patients. CONCLUSIONS: These findings demonstrate that moderate COVID-19 has a profound inflammation associated with severee ndotheliopathy and intense coagulation activation uncontrolled by TFPI. Attention should be paid to coagulopathy in COVID-19. Closely monitoring of coagulation and application of appropriate anticoagulation may improve the prognosis of moderate COVID-19 and to prevent the progression to severe COVID-19 disease.

Excessive blood loss due to trauma or major surgical intervention can be life threatening which necessitates rapid hemorrhage management for prevention of such bleeding related sufferings. Broad interest in developing new hemostatic technologies have been paid for bleeding control but none of them found completely satisfactory especially in terms of rapid clotting, absorbability, porosity, cost effectiveness and safety. To address these issues, a combination of active and passive hemostatic materials from biological sources could be a wise choice. Therefore, plant derived TEMPO-oxidized nanocellulose (TOCN)/biopolymer gelatin (G) sponge was successfully prepared in co-operation with intrinsic blood coagulation enzyme thrombin (Th) via freeze drying method and their application as rapid hemostatic dressing was investigated. Morphological and in vitro characteristics of the samples were evaluated where uniformity, porosity, swelling, degradation behavior had direct relationship with the percent gelatin incorporation. In vitro hemocompatibility and cyto-compatibility of these sponges were confirmed as well. Among the samples, TOCN 2.5 G-Th sponge exhibited excellent hemostatic effect, rapid absorbability, minimum clotting time (1.37 ± 0.152 min) and reduction of blood loss was ensured through rat liver punch biopsy model. The results demonstrated that, thrombin enhanced blood coagulation, platelet and red blood cell aggregation following application of biopolymer TOCN 2.5 G-Th sponge compared with samples devoid of thrombin. In short, the functional, cost effective and nontoxic sponge developed via facile preparation could potentially be used as an absorbable biomaterial to achieve immediate hemostasis. HighlightsPlant-derived TEMPO-oxidized nanocellulose (TOCN) and biopolymer gelatin (G) was successfully used to prepare a haemostatic sponge in combination with intrinsic blood coagulation enzyme thrombin (Th).The TG sponge combines the advantages of TOCN and Gelatin, exhibiting biocompatibility, biodegradability and superior blood-absorption performance.The TOCN 2.5G-Th sponge improves plasma absorption, red blood cell adhesion, aggregation, platelet adhesion and activation leading to enhanced hemostasis effect and shorter hemostasis time in vitro and in vivo.

Geriatric patients often have cardiovascular diseases that require differentiated perioperative management of hemostasis. The operation-related bleeding risk and the individual thromboembolism risk mutually influence each other, so that a differentiated preoperative assessment of the further prescription of coagulation-modulating medication is required. In many cases the active coagulation medication can be interrupted without replacement or continued unchanged. In cardiovascular diseases with antiplatelet medication, the preoperative risk-benefit assessment for most operations leads to the continuation of previous platelet aggregation inhibitor monotherapy; however, if there is a high risk of cardiovascular thromboembolism with dual platelet inhibition, the individual perioperative medication should be closely coordinated with a geriatrician or cardiologist.In most cases, the intake of vitamin K antagonists (VKA) can be preoperatively interrupted. In cases of high risk of thromboembolism, a temporary bridging with heparin must be carried out. The introduction of the four new direct oral antagonists (DOAC) has made the perioperative management of anticoagulation much easier. Bridging with heparin is not necessary. Perioperatively, only the dosage and timing of interruption of the DOACs have to be determined individually depending on the operative bleeding risk as well as the age, body weight and kidney function of the patient. If bleeding complications arise under the influence of the DOACs, antidotes are available for three of the four DOACs, which in acute cases can be used in addition to prothrombin complex concentrates and fresh frozen plasma to normalize coagulation.

Article, Blood Advances. 5(19): 3830-3838 (2021)

BACKGROUND Fresh frozen plasma (FFP) is used to treat coagulation disorders. Even though the activity of labile coagulation factors gradually decreases once thawed, it can be used up to 24 h after thawing, if stored properly. In this study, the level of coagulation factor activity was evaluated in thawed plasma during a 5 day storage period. MATERIALS AND METHODS This cross-sectional study was performed on 40 FFP units prepared in Yazd Blood Center. Samples were thawed in a waterbath for 20-30 min at 30-37°C and then stored in the hospital blood bank refrigerator. The level of fibrinogen concentration, as a stable factor and, coagulation factors V and VIII, as labile factors, were measured in the plasma immediately following the thawing process as well as 24 and 120 h after the process. Data analysis was performed using SPSS software 20. RESULTS The fibrinogen level remained stable for up to 24 h after thawing; after 120 h there was a 1.66% decrease with the mean level of 334.0 ± 53.3 mg/dl. The mean activity of factors V and VIII levels decreased by 12.3%, and 26% respectively over 120 h after thawing when compared to that after 24 h. A 120 h after thawing Factor V activity was above 70% in 87.5% of thawed plasmas and its mean activity was 81.6 ± 11.8. Factor VIII activity was above 70% in only 35% of thawed plasmas with the mean activity of 64.4 ± 17.2. CONCLUSION Thawed plasma can be used for up to 5 days in all therapeutic applications of FFP since it still has the essential hemostatic effects. However, in situations where higher levels of FVIII are needed, Thawed Plasma is not a suitable alternative. In such cases FFP, FVIII concentrate or cryoprecipitated antihemophilic factor should be used.

The risk of thromboembolism (TE) is increased in patients with inflammatory bowel disease (IBD), mainly due to an increased risk of venous TE (VTE). The risk of arterial TE (ATE) is less pronounced, but an increased risk of cardiovascular diseases needs to be addressed in IBD patients. IBD predisposes to arterial and venous thrombosis through similar prothrombotic mechanisms, including triggering activation of coagulation, in part mediated by impairment of the intestinal barrier and released bacterial components. VTE in IBD has clinical specificities, i.e., an earlier first episode in life, high rates during both active and remission stages, higher recurrence rates, and poor prognosis. The increased likelihood of VTE in IBD patients may be related to surgery, the use of medications such as corticosteroids or tofacitinib, whereas infliximab is antithrombotic. Long-term complications of VTE can include post-thrombotic syndrome and high recurrence rate during post-hospital discharge. A global clot lysis assay may be useful in identifying patients with IBD who are at risk for TE. Many VTEs occur in IBD outpatients; therefore, outpatient prophylaxis in high-risk patients is recommended. It is crucial to continue focusing on prevention and adequate treatment of VTE in patients with IBD.

Aims: Sapparin tablet, a medicine used for the treatment of blood diseases specially curing blood thickening or impure blood, liver disease in Mongolian Traditional Medicine. The objectives of the study were to determine total biological active substances and analyze the anticoagulation activity of the Sapparin. Study Design: Experimental study. Place and Duration of Study: Department of Chemistry and Technology and Department of Pharmacology, Institute of Traditional Medicine and Technology of Mongolia. Methodology: Quantitative determination of the total active constituents (phenolic, flavonoid, and carotinoids) of the methanol extracts of Sapparin was performed by using Folin-Ciocalteu reagent, aluminium chloride reagent by spectrophotometry. A totally of forty weighing between 220-250 gm were used. Effect of Sapparin was assessed on coagulation parameters following 7, 14, 21 and 28 days administration of 37 mg/kg, 56 mg/kg, 113 mg/kg to healthy rats. The blood coagulation parameters such as prothrombin time (PT) and the activated partial thromboplastin time (aPTT) were measured by means of Quick’s one-stage assay and modified aPTT assay respectively in the rats. Additionally, thrombin activity test was estimated in rats with PT assay using a hemagglutination analyzer. The levels of serum X and von Willebrand factor were measured in Sapparin and control groups by ELISA. Results: The total content of the phenols measured as 5.33±0.0005%, flavonoids as 12.95± 2.21% and carotinoids as 4.31±0.96%. There was significant increase in all assays except fibrinogen, prothrombin time, thrombin, aPTT. Sapparin treatment significantly reduced levels of serum X factor and von Willebrand factor was significantly decreased in rats. Conclusion: Results of this study suggest that Sapparin shows considerable anti-anticoagulant activity in animals and has potential to reduce cardiovascular morbidity and mortality.

Fibrinogen γ' accounts for 3% to 40% of plasma fibrinogen. Earlier studies indicated that fibrinogen γ' forms altered fibrin clots under static conditions, whereas clinically, altered plasma γ' levels are associated with arterial and venous thrombosis. However, the effects of static vs flow conditions on the role of γ′ throughout the pathophysiological range is unknown. This study explores the effects of γ' levels on clot formation and structure in static and flow conditions. Coagulation of plasma samples with low (n = 41; 3%), normal (n = 45; 10%), or high (n = 33; 30%) γ′ levels were compared with that of purified fibrinogen mixtures with increasing ratios of γ′ (3%, 10%, 30%). Clots were analyzed by confocal microscopy, permeation, turbidity, and lysis techniques. In a novel 2-step flow-perfusion model, fibrinogen-deficient plasma repleted with increasing ratios of γ′ (3%, 10%, 30%) or plasmas with low (n = 5, 3%) or high (n = 5, 30%) γ′ were flowed over preformed platelet aggregates at arterial (500 s−1) and venous (150 s−1) shear rates. Increasing γ′ percentages within the pathophysiological range (3%-30%) did not result in any change in clot-formation rates; however, it led to significantly higher clot density, thinner fibers, and slower lysis in static conditions. Under flow at arterial shear, high γ′ (30%) led to faster (+44.1%-75.3%) and increased (+104%-123%) fibrin deposition, with clots exhibiting a larger volume (+253%-655%) and height (+130%-146%). These trends were magnified at venous shear. Overall, our findings demonstrate the significant impact of pathophysiological fibrinogen γ′ levels on clot structure and provide new flow-dependent mechanisms to explain how γ′ increases thrombosis risk.

Cationic carbon dots (CCDs) are a promising alternative to gene-delivery systems, and good biosafety levels are crucial for their in vivo use. In this study, spherical and monodispersed CCDs with an average surface potential of +28.7 mV were prepared using sucrose and glutamate (denoted SG-CCDs) using a one-pot autoclave-assisted method. Molecular interactions between the SG-CCDs and four major human serum proteins (albumin, immunoglobulin G, fibrinogen, and transferrin) were investigated. The results were further verified on human serum, and the effect of the SG-CCDs on in vitro blood coagulation was examined. The results showed that the fluorescence of human serum was clearly quenched by the SG-CCDs through a dynamic collision mechanism. Moreover, SG-CCDs at a concentration of 20 μM exhibited minor effects on the secondary structure of human serum. The activated partial thromboplastin and prothrombin time as well as the fibrinogen concentration were not changed, indicating that the SG-CCDs did not interfere with the coagulation process. This study provided an understandable background on the behaviour of CCDs in clinical applications.

Contact activation refers to the process of surface-induced activation of factor XII (FXII), which initiates blood coagulation and is captured by the activated partial thromboplastin time (aPTT) assay. Here, we show the mechanism and diagnostic implications of FXII contact activation. Screening of recombinant FXII mutants identified a continuous stretch of residues Gln317–Ser339 that was essential for FXII surface binding and activation, thrombin generation and coagulation. Peptides spanning these 23 residues competed with surface-induced FXII activation. Although FXII mutants lacking residues Gln317–Ser339 were susceptible to activation by plasmin and plasma kallikrein, they were ineffective in supporting arterial and venous thrombus formation in mice. Antibodies raised against the Gln317–Ser339 region induced FXII activation and triggered controllable contact activation in solution leading to thrombin generation by the intrinsic pathway of coagulation. The antibody-activated aPTT allows for standardization of particulate aPTT reagents and for sensitive monitoring of coagulation factors VIII, IX, XI., Blood coagulation is started by contact to surfaces and this is the principle for a commonly used diagnostic clotting test, aPTT. Here, the authors identify the structure in coagulation factor XII that initiates surface-driven coagulation and use the information to develop improved aPTT assays.

BACKGROUND Coagulation factor IX (FIX) is a serine protease zymogen involved in the intrinsic blood coagulation pathway, and its deficiency causes hemophilia B. Zebrafish has three f9 genes, and the ortholog to human F9 is unknown. OBJECTIVE To identify the zebrafish ortholog to F9 using sequence analysis and piggyback knockdown technology. METHODS Gene and protein sequence analysis for three f9 genes, f9a, f9b, and f9l, present in the zebrafish genome was performed. In vivo and in vitro assays after knockdown of each gene and immunodepletion using specific antibodies were carried out. RESULTS Sequence analysis revealed that f9a and f9b are similar to human F9, whereas f9l is similar to human F10. RNA analysis showed an age-dependent increase in expression of all three genes. Zebrafish f9a gene knockdown and Fixa immunodepletion prolonged kinetic partial thromboplastin time (kPTT), whereas f9l knockdown and Fixl immunodepletion prolonged kPTT, kinetic prothrombin time, and kinetic Russell viper venom activation time. Laser-assisted venous thrombosis increased time to occlusion after f9a and f9l knockdown and antibody inhibition of Fixa and Fixl. Further, analysis of plasma proteins by mass spectrometry and immunohistochemistry detected all three proteins. CONCLUSIONS Our findings suggest that zebrafish f9a has functional activity similar to human F9. Fixl is functionally similar to Fx. The age-dependent increases of these factors are comparable to those observed in mice and humans. Thus, the zebrafish model could be used to study factors involved in increasing f9a expression during aging. It could also be used to test whether normal human Factor IX and Factor IX Leyden promoter work in zebrafish background.

The aim of this study was to analyze the role of blood biomarkers regarding preoperative inflammation and coagulation in predicting the postoperative in-hospital mortality of patients with type A acute aortic dissection (AAD). A total of 206 patients with type A AAD who had received surgical treatment were enrolled in this study. Patients were divided into two groups: the death group (28 patients who died during hospitalization) and the survival group (178 patients). Peripheral blood samples were collected before anesthesia induction. Preoperative levels of D-dimer, fibrinogen (FIB), platelet (PLT), white blood cells (WBC) and neutrophil (NEU) were compared between the two groups. Univariable and multivariable logistic regression analysis were utilized to identify the independent risk factors for postoperative in-hospital deaths of patients with type A AAD. Receiver operating characteristic (ROC) curve were used to analyze the predictive value of these indices in the postoperative in-hospital mortality of the patients. Univariable logistic regression analysis showed that the P values of the five parameters including D-dimer, FIB, PLT, WBC and NEU were all less than 0.1, which may be risk factors for postoperative in-hospital deaths of patients with type A AAD. Further multivariable logistic regression analysis indicated that higher preoperative D-dimer and WBC levels were independent risk factors for postoperative in-hospital mortality of patients with type A AAD. ROC curve analysis indicated that application of combining FIB and PLT could improve accuracy in prediction of postoperative in-hospital mortality in patients with type A AAD. Both preoperative D-dimer and WBC in patients with type A AAD may be used as independent risk factors for the postoperative in-hospital mortality of such patients. The combination of FIB and PLT may improve the accuracy of clinical prognostic assessment.

New modified coagulation factor VIII (FVIII) and factor IX (FIX) products have been designed to improve the treatment of individuals with hemophilia A and B by increasing the interval between dosing. Although these FVIII and FIX molecules have been structurally modified to improve the circulation time, the changes have also influenced their behavior in functional assays in comparison with traditional plasma-derived or recombinant coagulation factors. The assignment of potencies for these products can be problematic because discordance in factor activity values between the commonly used one-stage clotting (OC) and chromogenic substrate (CS) assays is often observed. Discrepancies in potency assay values also exist when different assay kits and reagents are used in the same assay type. Ideally, all FVIII and FIX products should be calibrated against the World Health Organization (WHO) International Standards (IS) because the assignment of potencies in International Units (IU) helps maintain treatment tradition and meaningful references for manufacturers, patients, and clinicians. The discrepant measurements, attributed to the modified structural and functional properties of these products, are manifested in their lack of commutability with the WHO IS for FVIII or FIX. Herein, we discuss the considerations upon which an assay is chosen for potency assignment and post-administration monitoring of a new factor product, which include the validity of the assay calibrated with the IS, the meaning of the potency values in IU, standards of care for patients, clinical relevance between the assigned potency value and recovery value from clinical labs, and patient safety.

Background Hyperthyroid cats might have a predisposition to arterial thrombus formation. The mechanism for thrombogenesis currently is unknown but could be associated with systemic hypercoagulability as seen in hyperthyroid humans. Objective Our purpose was to evaluate markers of hemostasis in hyperthyroid cats compared to healthy cats, and in hyperthyroid cats before and after radioactive iodine treatment (RIT). Animals Twenty‐five cats with hyperthyroidism and 13 healthy euthyroid cats >8 years of age. Methods Prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen concentration, antithrombin (AT), D‐dimers, thrombin‐antithrombin complexes (TAT), von Willebrand Factor antigen (vWF : Ag), and activity of factors VIII and IX were measured. An echocardiogram was performed in all cats. Hemostatic markers and echocardiogram were evaluated again 6 to 9 months after successful RIT in 7 cats. Results Hyperthyroid cats had higher fibrinogen concentration (P

Coronavirus disease 2019 (COVID-19) is a pandemic syndrome caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. SARS-CoV-2 infection induces a process of inflammation and thrombosis supported by an altered platelet activation state. This platelet activation is peculiar being characterized by the formation of platelet-leukocytes rather than platelet–platelet aggregates and by an increased procoagulant potential supported by elevated levels of TF positive platelets and microvesicles. Therapeutic strategies targeting, beyond systemic inflammation (i.e. with tocilizumab, an anti interleukin-6 receptor), this state of platelet activation might therefore be beneficial. Among the antithrombotic drugs proposed as candidates to treat patients with SARS-CoV-2 infection, antiplatelet drugs, such as aspirin are showing promising results.

Background COPD patients have an increased risk of cardiovascular disease and venous thromboembolism. Methods This study aimed to investigate whether patients with stable COPD have a prothrombotic state compared to COPD-free smokers. We conducted an observational study comparing levels of: D-dimers, INR, aPTT, coagulation factors; fibrinogen, FII, FV, FVII, FVIII, FIX, FX and coagulation inhibitors; protein S, proteins C and antithrombin between stable COPD patients and control subjects. Results A total of 103 COPD patients and 42 controls with similar age, sex, current smoking status, comorbidity burden and cardiovascular risk met the inclusion criteria. Compared to controls, COPD patients had higher levels of D-dimers (median (interquartile range): 360 (230–600) ng·mL−1 versus 240 (180–400) ng·mL−1, p=0.001), fibrinogen (mean±sd: 399±82 mg·dL−1 versus 346±65 mg·dL−1, p, Patients with stable COPD exhibit increased levels of key coagulation factors and decreased levels of coagulation inhibitors, namely protein S and antithrombin, compared to COPD-free smokers, indicating a prothrombotic state in stable COPD https://bit.ly/2VmR1PP

Background Double-filtration plasmapheresis (DFPP), a selective therapeutic apheresis, can deplete pathogenic antibodies/substances, but also important coagulation factors. Aim To determine if the use of a separator filter with different characteristics (CascadefloEC-50 W) as compared to the reference filter (PlasmafloOP-08 W) is as efficient in terms of immunoglobulin loss, but can reduce coagulation factor losses and have similar tolerability. Patients/methods This is a single-center prospective study including 14 patients divided into two groups (7 each): that is, group1 = CascadefloEC-50 W and group2 = PlasmafloOP-08 W. We measured immunoglobulins, lipid profiles, blood-cell counts, hemostasis (prothrombin time, activated partial thromboplastin time), coagulation factors, and natural anticoagulants at before and after the first DFPP-session. Results In group 1, the loss of coagulation factors was significantly reduced as compared to group 2 for proteins with a molecular weight of >150 kDa: there was, respectively, an average decrease of 70% vs 31% for fibrinogen (P = 0.004), 66% vs 21% for factor V (P = 2.16e-07), 60% vs 32% for factor XI (P = 6.96e-06), 75% vs 17% for XIII-antigen (P = 0.0002), and 47% vs 0% for VWF-antigen(P = 0.02). The decrease in post-session IgG was, on average, 45% in group 1 and 50% in group 2 (P = 0.13). Those results remained significant even when adjusted to the treated-plasma volume and the pre-DFPP factor values. Conclusion DFPP, using a CascadefloEC-50W as a first-filter, reduces efficiently IgGs similarly to PlasmafloOP-08W but spares clotting factors.

Background: Patients with platelet dysfunction disorders present with a variety of mucocutaneous bleeding symptoms including easy bruising, frequent epistaxis, bleeding gums upon tooth brushing and for women, heavy menstrual bleeding. Available laboratory assays to evaluate platelet function include the platelet function analyzer (PFA) and in larger centers with coagulation laboratories, light transmission platelet aggregometry (LTA) analyses. Both assays are known to have a number of limitations, especially in the diagnosis of platelet delta granule storage pool deficiency (gamma-SPD).Gamma-SPD is an underdiagnosed condition caused by decreased numbers of platelet dense granules (DGs) and is best diagnosed by electron microscopy (EM). Patients with platelet gamma-SPD have a decreased response to low levels of the agonist adenosine diphosphate (ADP) in the second wave of light transmittance with LTA or decreased ADP secretion by fluorescence lumiaggregometry. There are few reports that have evaluated patients withgamma-SPD and their respective LTA results. One report published in 1987 described normal LTA assays in 23% of patients with gamma-SPD; a more recent report described LTA as having the sensitivity to detect only about 52% of patients with gamma-SPD. The purpose of our study was intended to review the LTA and EM results of patients suspected of having a platelet function disorder at our institution for comparison with previously published studies. Methods: Our study included 344 patients who had been evaluated by both LTA and whole mount EM. Aggregometry utilized five agonists: ADP, epinephrine, collagen, arachidonic acid, and ristocetin. DGs were enumerated in 100 whole-mounted platelets to determine a mean number of dense granules per platelet (DGs/PL). Results: Seventy-seven percent of our patients were found to have gamma-SPD (264/344); 68% (179/264) of these subjects had an abnormal platelet LTA. Thirty-two percent (85/264) of our patients had normal LTA results but were found to have gamma-SPD with a mean of 2.54 ± 0.15 DG/PL (normal = 4 - 6 DG/PL). Conclusion: These data confirm previous reports suggesting the utilization of LTA alone in patients with histories of unexplained bleeding may miss the diagnosis of platelet gamma-SPD. It is, therefore, prudent to assess platelet DG number by EM, especially if platelet LTA assessment is normal. J Hematol. 2021;10(4):196-201 doi: https://doi.org/10.14740/jh832