Hemostatic Potential Research Articles

Технология низкочастотной пьезотромбоэластографии в мониторинге противотромботической терапии

Представлена информация о новой технологии оценки гемостатического потенциала цельной крови в процессе коагуляции — низкочастотной пьезотромбо-эластографии, позволяющей проводить интегративную оценку его характеристик. На примере оценки эффектов антиагрегантов (ингибитора ЦОГ-1 — ацетилсалициловой кислоты, блокатора АДФ-рецепторов тромбоцитов — клопидогрела) и антикоагулянтов (нефракционированного гепарина — гепарината натрия, низкомолекулярного гепарина — эноксапарина) продемонстрированы возможности оперативной и персонифицированной оценки их фармакодинамики. Получены новые данные о специфических и плейотропных эффектах противотромботических лекарственных средств. Специфическим ответом гемостатического потенциала в ответ на прием антиагрегантов является снижение агрегационной активности форменных элементов крови, проявляющееся повышением суспензионной стабильности крови и снижением интенсивности контактной коагуляции. Плейотропные эффекты антиагрегантных препаратов проявляются тенденцией к изменению гемостатического потенциала в сторону структурной и хронометрической гипокоагуляции. По результатам оценки фармакодинамики антикоагулянтов выявлено время их максимального действия на гемостатический потенциал, а также длительность антикоагулянтного эффекта. Специфическим ответом гемостатического потенциала после введения антикоагулянтов является развитие структурной и хронометрической гипокоагуляции, реализующейся за счет снижения протеолитического этапа фибриногенеза и активности процессов латеральной сборки фибрина.

Thromboelastometry Velocity Curve Predicts Blood Product Transfusion and Mortality Within the First 24 Hours in Severely Injured Trauma Patients

SESSION TITLE: Hematology/Oncology in the ICU SESSION TYPE: Original Investigation Slide PRESENTED ON: Monday, October 30, 2017 at 04:30 PM - 05:30 PM PURPOSE: The processes leading to uncontrolled bleeding after trauma are complex and heterogeneous. Thrombin generation (TG) is pivotal for an adequate hemostatic response but burdensome to assess in emergency settings. Instead, thromboelastometry (TEM), is an increasingly used point of care test for trauma patients care. TEM estimates the first derivative of the clot growth velocity curve (V-curve), which has a remarkable similarity with the endogenous thrombin potential trace and is highly correlated to TG determination by thrombin/antithrombin complex. TEM V-curve describes the maximum speed of clot formation (MaxV), the time to reach MaxV (MaxV-t) and the area under the curve (AUC). The study objective is to evaluate whether the TEM V-curve parameters (MaxV, MaxV-t, and AUC) can predict blood transfusion and mortality in the first 24h post-injury in adult trauma patients. METHODS: Retrospective cohort study of severely injured patients admitted to a Level I Trauma Center. Severity was defined as any abbreviated Injury Score (AIS) >2. Demographic data including mortality, conventional coagulation tests, rotational thromboelastometry on admission, and the total amount of blood products transfused in the first 24h were collected. With a linear regression model, we examined the effect of the V-curve parameters and the total number of blood products in 24h, adjusting for systolic pressure (SBP), temperature, injury severity score (ISS), base excess (BE), hemoglobin(HB), INR, fibrinogen, aPTT, and platelets levels. With a logistic regression model, we assessed the effect of V-curve parameters in 24h mortality, adjusting for age, AIS head, ISS, and BE. RESULTS: 550 patients were included with median ISS 19 and a median age of 43 years. The maximum speed of clot formation (MaxV) was associated with the number of units of blood product transfused in 24h of injury; for every unit decrease in MaxV (mm/min) there was an average increase of 0.7U transfused in 24h (95%CI 0.29-1.13, p= <0.0001) after adjusting for SBP, temperature, ISS, BE, hemoglobin, INR, fibrinogen, aPTT, platelets levels at admission, and the other V-curve parameters (MaxV-t, AUC). Other parameters affecting 24h blood transfusion were SBP (p= <0.0001), AUC (p= 0.037), BE (p= <0.0001). Time to reach MaxV (MaxV-t) not associated with blood transfusion. In a logistic regression model, MaxV was was associated with 24h mortality (OR 1.3, 95%CI 1.12-1.51, p= 0.0005) after adjusting for age, AIS head, ISS, and BE. Also, age(p= 0.0068), traumatic brain injury(p= 0.0027), and BE(p= 0.0033) were associated with 24h mortality. CONCLUSIONS: Our study is among the first to demonstrate an association between the TEM V-curve and clinical outcomes in trauma. Reductions in MaxV were associated with increased transfusion requirements and mortality in the initial 24h following injury, adding to the understanding of early trauma coagulopathy, that the velocity of clot growth rather than the total amount of thrombin impacts the clinical consequences significantly. CLINICAL IMPLICATIONS: TEM V-curve parameters evaluate the hemostatic potential, understanding the thrombin generation and clot formation dynamics in trauma patients with significant hemorrhage are crucial for developing adequate hemostatic strategies. DISCLOSURE: The following authors have nothing to disclose: Johana Gomez Builes, Sandro Rizoli, Andrew Baker No Product/Research Disclosure Information

Different chemical groups modification on the surface of chitosan nonwoven dressing and the hemostatic properties

The hemostatic properties of surface modified chitosan nonwoven had been investigated. The succinyl groups, carboxymethyl groups and quaternary ammonium groups were introduced into the surface of chitosan nonwoven (obtained NSCS, CMCS and TMCS nonwoven, respectively). For blood clotting, absorbance value (0.105±0.03) of NSCS1 nonwoven was the smallest (CS 0.307±0.002, NSCS2 0.148±0.002, CMCS1 0.195±0.02, CMCS2 0.233±0.001, TMCS1 0.191±0.002, TMCS2 0.345±0.002), which indicated the stronger hemostatic potential. For platelet aggregation, adenosine diphosphate agonist was added to induce the nonwoven to adhered platelets. The aggregation of platelet with TMCS2 nonwoven was highest (10.97±0.16%). Further research of blood coagulation mechanism was discussed, which indicated NSCS and CMCS nonwoven could activate the intrinsic pathway of coagulation to accelerate blood coagulation. NSCS1 nonwoven showed the shortest hemostatic time (147±3.7s) and the lowest blood loss (0.23±0.05g) in a rabbit ear artery injury model. These results demonstrated that these surface modified chitosan nonwoven dressings could use as a promising hemostatic intervention, especially NSCS nonwoven dressing.

The pharmacokinetics and pharmacodynamics of single-dose and multiple-dose recombinant activated factor VII in patients with haemophilia A or B.

To compare the pharmacokinetics (PK) of rFVIIa after 3×90μgkg-1 doses vs a single dose (270μgkg-1 ) in haemophilia patients and to evaluate TEG/TGA results postdosing to determine how these assays relate to PK findings. Patients in this open-label, single-centre, randomized, crossover trial received one injection of 270μgkg-1 rFVIIa crossed over with three injections of 90μgkg-1 rFVIIa in a non-bleeding state. For TEG, kaolin and tissue factor were used as activators; TGA was performed on frozen platelet-rich and platelet-poor plasma, with and without corn trypsin inhibitor. FVIIa activity was evaluated using in vivo samples. TGA showed a dose-dependent effect of rFVIIa on thrombin generation; TEG revealed lower dose-dependent effects. Both showed some differences between single-/multiple-dose rFVIIa; both supported the PK findings. While TEG and TGA are not yet clinically predictive, both supported the PK results. Data suggest that, while a single dose of 270μgkg-1 rFVIIa provides slightly higher haemostatic potential than the multiple-dose regimen of 3×90μgkg-1 , the latter results in prolonged activity levels compared with a higher single dose.

Analysis of the hemostatic potential of modern topical sealants on arterial and venous anastomoses: an experimental porcine study

One of the widely accepted adjunctive agents in the variety of surgical modalities are sealants. Our study aim was to compare four commonly used modern sealants in a standardized experimental setting to assess their feasibility, and hemostatic efficacy in vascular anastomosis. Forty landrace pigs (weight: 24.7 ± 3.8 kg) were randomized into the control (n = 8) and four sealant groups; TachoSil® (n = 8), Tissucol Duo® (n = 8), Coseal® (n = 8), and FloSeal® (n = 8). After doing a portal vein end-to-end anastomosis as well as stitches of aortic incision, the sealants were applied on anastomotic site. The control group was left intact. In portal vein anastomosis, the sealants led to a complete hemostasis significantly better than control group. The mean of blood loss was also significantly reduced. In successful subgroups, there was a difference in the mean-time to reach complete hemostasis ranging from 15 s in Coseal® to 76 s in FloSeal® group (p < 0.05). In aortotomy experiments, except Tissucol Due®, which had insufficient hemostasis, other sealants led to a complete hemostasis. The mean blood loss was significantly reduced in sealants groups as well. The four sealants are effective in reducing the suture-hole bleeding in portal vein anastomosis. However, the hemostatic potential is heterogeneous among sealants. This means that "one-size-fits-all" approach is not appropriate for application of sealants in diversity of vascular surgery and it should be based on the type and the severity of injury and the structure of tissue. Comparison of hemostasis efficacy of four modern sealants (TachoSil®, Tissucol Duo®, Coseal®, and FloSeal®) in vascular anastomosis in porcine model. The figures below show the total blood loss (g) in the control and sealant groups after aortotomy (left) and portal vein anastomosis (right). The mean of blood loss decreased significantly by the usage of sealants in both experiment groups as compared to control group (*: p < 0.05; sealant groups vs. control group). 1. The right column shows the mean of blood loss (g) in all experiments in each group. 2. The middle column presents the subgroup with unsuccessful hemostasis at the end of observation time (Tmax = 20 sec. for aortotmy and 300 sec. for portal vein anastomosis). 3. The left column shows mean of total blood loss in subgroups with successful hemostasis during observation time (20 sec for aortotomy and 300 sec for portal vein).

An in silico and in vitro approach to elucidate the impact of residues flanking the cleavage scissile bonds of FVIII.

Coagulation Factor VIII is activated by an ordered limited thrombin proteolysis with different catalytic efficiency at three P1 Arginine residues: Arg759> Arg1708>Arg391, indicating the flanking residues of the latter to be less optimal. This study aimed to investigate, in silico and in vitro, the impact of possessing hypothetically optimized residues at these three catalytic cleavage sites. The structural impact of the residues flanking Arginine cleavage sites was studied by in silico analysis through comparing the cleavage cleft of the native site with a hypothetically optimized sequence at each site. Moreover, recombinant FVIII proteins were prepared by replacing the sequences flanking native thrombin cleavage sites with the proposed cleavage-optimized sequence. FVIII specific activity was determined by assessing the FVIII activity levels in relation to FVIII antigen levels. We further investigated whether thrombin generation could reflect the haemostatic potential of the variants. Our in silico results show the impact of the residues directly in the cleavage bond, and their neighboring residues on the insertion efficiency of the loop into the thrombin cleavage cleft. Moreover, the in vitro analysis shows that the sequences flanking the Arg1708 cleavage site seem to be the most close to optimal residues for achieving the maximal proteolytic activation and profactor activity of FVIII. The residues flanking the scissile bonds of FVIIII affect the cleavage rates and modulate the profactor activation. We were able to provide insights into the mechanisms of the specificity of thrombin for the P1 cleavage sites of FVIII. Thus, the P4-P2´ residues surrounding Arg1708 of FVIII have the highest impact on rates of thrombin proteolysis which contributes to thrombin activation of the profactor and eventually to the thrombin generation potential.

The role of low-frequency piezothromboelastography in the comprehensive evaluation of hemostasis&#x0D; in coronary bypass surgery with cardiopulmonary bypass

To monitor the hemostatic system during platelet concentrate transfusions using low-frequency piezothromboelastography in patients with coronary bypass surgery given aspirin therapy. The study involved 148 patients with coronary bypass surgery, with 76 ones undergoing intraoperative transfusion of platelet concentrate and 72 treated without transfusion. The control group consisted of 20 healthy individuals. In the perioperative period indicators of vascular-platelet, coagulation, anticoagulant and fibrinolytic components of hemostasis were evaluated by low-frequency pezotromboelastography using the ARP-01M "Mednord" hardware and software system (Russia). It was shown that the antiplatelet effect of aspirin in the preoperative period was manifest as inhibition of the initial stage of blood coagulation accompanied by increased thrombin potential, the total gain of anticoagulant and fibrinolytic activity of the blood. Heart-lung bypass was accompanied by structural and chronometric anticoagulation, reduction of anticoagulation and increase of blood fibrinolytic activity. In the postoperative period, structural and chronometric anticoagulation was more pronounced in patients who did not undergo transfusion of platelet concentrate than in the group of patients with transfusion. Donor platelets further increased the blood hemostatic potential by neutralizing the antiplatelet effect of aspirin. Perioperative thrombohemorrhagic complications were absent in both groups. Low-frequency piezothromboelastography provides a tool for real-time monitoring the functional state of hemostasis system. Transfusion of platelet concentrate is not advisable given that the thrombin potential is preserved as confirmed by the results of low-frequency piezothromboelastography.

Use of thrombin generation test for monitoring hemostasis in coronary bypass surgery.

To evaluate the parameters of the thrombin generation test (TGT) in coronary artery disease (CAD) patients on prolonged aspirin therapy during on-pump coronary artery bypass grafting (CABG) after donor platelet concentrate transfusion. A total of 148 patients with CAD on prolonged aspirin therapy (75–100 mg/day) who have undergone elective on-pump CABG were consecutively included in the study. Patients were divided randomly into two groups. Group 1 (n = 76) received donor platelet transfusions after cardiopulmonary bypass, whereas Group 2 (n = 72) did not. TGT parameters were measured using an analyzer at pre-, intra-, and early postoperative periods. Activation of the endogenous thrombin potential was observed in patients on prolonged aspirin therapy in the pre- and intraoperative periods, as confirmed by high peak thrombin and increased velocity index. The activation time of the prothrombinase complex and thrombin generation time were greater than the control group. The blood hemostatic potential in patients who did not receive transfusions in the early postoperative period decreased up to the level of the control group in the extended time parameters. Hemostatic potential in plasma in patients on aspirin was preserved. Given the laboratory test results and clinical data, platelet concentrate transfusion is unnecessary for prevention.

Potential role of platelet-leukocyte aggregation in trauma-induced coagulopathy: Ex vivo findings.

Platelet dysfunction has been identified as an important contributor of trauma-induced coagulopathy, but the underlying mechanism still remains to be elucidated. Trauma-associated proinflammatory stimuli strongly activate leukocytes, which in turn bind activated platelets. Therefore, we investigated the role of platelet-leukocyte aggregation (PLA) as a potential feature of trauma-induced platelet dysfunction. Whole blood from 10 healthy donors was exposed to selective and collective platelet and leukocyte agonists in order to simulate differential states of activation. PLA formation and CD11b expression as a measure of leukocyte activation were determined by flow cytometry. Platelet-mediated hemostatic function was measured by thromboelastometry (ROTEM) and impedance aggregometry (Multiplate). Activation of platelets and leukocytes was associated with diminished platelet-mediated hemostatic potential. Aggregation of platelets with monocytes rather than granulocytes resulted in a reduction of hemostatic function, as indicated by an impaired responsiveness in platelet aggregometry and a reduction of thromboelastometric maximum clot firmness. This finding was irrespective of CD11b expression and was not paralleled by a reduction of measurable platelet counts. PLA formation occurs primarily between monocytes and activated platelets and is associated with impaired platelet-mediated hemostatic function. PLA formation was not paralleled by a reduction in platelet complete blood counts.

Comparing thrombin generation in patients with hemophilia A and patients on vitamin K antagonists

Essentials It is unknown if hemophilia patients with atrial fibrillation need anticoagulation. Endogenous thrombin potentials (ETP) in hemophilia patients and patients on coumarins were compared. Severe hemophilia patients had comparable ETP to therapeutic international normalized ratio (INR). In non-severe hemophilia, 33% had higher ETP than therapeutic INR and may need anticoagulation. Click to hear Dr Negrier's perspective on global assays for assessing coagulation SUMMARY: Background It is unknown whether patients with hemophilia A with atrial fibrillation require treatment with vitamin K antagonists (VKAs) to the same extent as the normal population. Objective To compare hemostatic potential in hemophilia patients and patients on VKAs using thrombin generation (TG). Methods In this cross-sectional study, TG, initiated with 1pM tissue factor, was measured in 133 patients with severe (FVIII < 1%, n = 15) and non-severe (FVIII 1-50%, n = 118) hemophilia A, 97 patients on a VKA with an international normalized ratio (INR) ≥ 1.5 and healthy controls. Endogenous thrombin potential (ETP) (nm*min) was compared according to FVIII level (< 1%, 1-19% and 20-50%) with healthy controls and patients with sub-therapeutic INR (1.5-1.9) and therapeutic INR (≥ 2.0). Medians and interquartile ranges (IQRs) were calculated. Results Compared with healthy controls (898 [IQR 803-1004]), both hemophilia patients and patients on VKAs had lower median ETPs at 304 (196-449) and 176 (100-250), respectively. ETP was quite similar in severe hemophilia patients (185 [116-307]) and patients with a therapeutic INR (156 [90-225]). Compared with patients with therapeutic INR, ETP in patients with FVIII 1-19% and patients with FVIII 20-50% was higher at 296 (203-430) and 397 (219-632), respectively. All patients with therapeutic INR had an ETP < 400. Considering this threshold, 93% of severe hemophilia patients, 70% of patients with FVIII 1-19% and 52% of patients with FVIII 20-50% had an ETP < 400. Conclusion In severe hemophilia patients, TG was comparable to that in patients with a therapeutic INR. In one-third of non-severe hemophilia patients, TG was higher. These results suggest that anticoagulation therapy should be considered in a substantial proportion of non-severe hemophilia patients.

Abstract 147: Plasma Factor XIII Binding to Cold-stored Platelets Results in Increased Fibrin Crosslinking and Clot Strength

Currently, platelets (PLTs) stored at room temperature (RT) for 5-7 days with gentle agitation are exclusively used for transfusion although FDA recently clarified that apheresis PLTs stored at 4°C for up to 72 hours may be used for treating active hemorrhage. We have demonstrated that cold (4C) storage of PLT is an attractive alternative to RT storage since it better preserves the PLT metabolic reserves, in vitro responses to agonists of activation, aggregation and physiologic inhibitors, as well as adhesion to thrombogenic surfaces. In this study, we tested the hypothesis that 4C-stored PLT will form clots with mechanical strength superior to those from RT-stored PLT due to higher hemostatic potential. From rheological measurements, we observed that the clots formed from 5 day 4C-stored PLTs are significantly stiffer (elastic modulus) and stronger (critical stress) than those formed from RT-stored PLT but comparable to fresh PLT (Fig. A). We also observed from ultrastructural microscopy that the fibrin fibers in clots from cold-stored PLT were thinner with more branch points than those from RT-stored PLTs, indicating the presence of increased crosslinks (Fig. B, C). Finally, molecular analysis revealed an increase FXIII transglutaminase activity due to the binding of plasma FXIII/fibrinogen to the surface of 4C-stored PLTs (Fig D).In conclusion, we have shown that cold-induced plasma FXIII binding to PLT surface result in increased fibrin crosslinking and enhanced clot strength. Our data, together with the benefit of reduced risk of late thrombosis due to their rapid clearance in vivo , underscores the consideration of 4C-stored PLT for acute response to hemorrhage. Figure 1 (A) RT-stored platelets form clots with less stiffness (n=5); (B) Representative SEM images of clots (n=3) (C) Clots from 4C-stored platelets have higher cross-linking density (n=5) (D) In FXIII-deficient plasma, 4C-stored platelets form clots with higher stiffness than fresh platelets (n=4)

Rational and timely haemostatic interventions following cardiac surgery - coagulation factor concentrates or blood bank products

BackgroundCardiac surgery may cause a serious coagulopathy leading to increased risk of bleeding and transfusion demands. Blood bank products are commonly first line haemostatic intervention, but has been associated with hazardous side effect. Coagulation factor concentrates may be a more efficient, predictable, and potentially a safer treatment, although prospective clinical trials are needed to further explore these hypotheses. This study investigated the haemostatic potential of ex vivo supplementation of coagulation factor concentrates versus blood bank products on blood samples drawn from patients undergoing cardiac surgery. Methods30 adults were prospectively enrolled (mean age=63.9, females=27%). Ex vivo haemostatic interventions (monotherapy or combinations) were performed in whole blood taken immediately after surgery and two hours postoperatively. Fresh-frozen plasma, platelets, cryoprecipitate, fibrinogen concentrate, prothrombin complex concentrate (PCC), and recombinant FVIIa (rFVIIa) were investigated. The haemostatic effect was evaluated using whole blood thromboelastometry parameters, as well as by thrombin generation. ResultsImmediately after surgery the compromised maximum clot firmness was corrected by monotherapy with fibrinogen or platelets or combination therapy with fibrinogen. At two hours postoperatively the coagulation profile was further deranged as illustrated by a prolonged clotting time, a reduced maximum velocity and further diminished maximum clot firmness. The thrombin lagtime was progressively prolonged and both peak thrombin and endogenous thrombin potential were compromised. No monotherapy effectively corrected all haemostatic abnormalities. The most effective combinations were: fibrinogen+rFVIIa or fibrinogen+PCC. Blood bank products were not as effective in the correction of the coagulopathy. ConclusionCoagulation factor concentrates appear to provide a more optimal haemostasis profile following cardiac surgery compared to blood bank products.

Increased Coagulation and Decreased Fibrinolysis as Measured with Overall Hemostatic Potential Are Dependent on BMI and Not Associated with PCOS.

Overall hemostatic potential (OHP) captures all factors that affect coagulation and fibrinolysis cascade. It has not yet been assessed in polycystic ovary syndrome (PCOS). The aim of the study was to identify the relationship of OHP with a syndrome per se and body mass index (BMI). In 90 women with PCOS aged 30.9 ± 8.1 years (50 obese, 13 overweight, and 27 lean) and 21 healthy age-matched controls (11 obese and 10 lean), OHP with overall coagulation potential (OCP) and overall fibrinolytic potential (OFP) was determined spectrophotometrically. OFP was calculated. OHP increased with BMI in PCOS (9.6 ± 2.3 in lean, 12.5 ± 5.1 in overweight, and 15.5 ± 3.8 Abs-sum in obese) and in controls (9.1 ± 1.0 in lean and 17.3 ± 4.6 Abs-sum in obese). There was significant difference between lean and obese PCOS (P < 0.001) and between lean and obese controls (P < 0.001). OCP also increased with BMI in PCOS (P < 0.001 for lean vs. obese) and in controls (P < 0.001 for lean vs. obese). OFP decreased with BMI in PCOS (P < 0.001 for obese vs. overweight vs. lean) and in controls (P < 0.001 for obese vs. lean). OHP in healthy obese and obese PCOS did not differ significantly, while OHP for healthy obese was increased in comparison to overweight and lean PCOS (P < 0.001). PCOS was not associated with increased OHP compared with BMI and age-matched controls. However, increase in OHP was positively associated with BMI in PCOS and healthy women.

The impact of frailty on coagulation and responses to warfarin in acute older hospitalised patients with atrial fibrillation: a pilot study.

The evidence on coagulation changes with frailty is not consistent and clinical studies suggest that frail older people may be at an increased risk of bleeding complications with anticoagulant therapy. This study aims to assess the impact of frailty on coagulation function and on response to warfarin. Inpatients aged over 65years with atrial fibrillation (AF) were recruited. Frailty was determined using the Reported Edmonton Frail Scale. The Overall Haemostatic Potential (OHP) and Calibrated Automated Thrombogram (CAT) were used to globally assess coagulation function. Data of 95 participants were analysed, mean age 85.5 ± 6.2, 40% female, and 50.5% frail. Among participants not on anticoagulants (N = 36), there was an increased fibrin generation and decreased thrombin generation compared to the local established normal ranges in young healthy volunteers; the frail had significantly reduced fibrin generation compared to the non-frail. In the warfarin-treated group (N = 59), there was no difference on coagulation profiles between the frail and the non-frail from any of the coagulation tests. In this cohort of acute hospitalised patients with AF, the observed decreased fibrin generation in the frail may reflect decreased acute phase response as suggested with the lower plasma fibrinogen in that group. There was no difference in coagulation profiles between the frail and the non-frail amongst those taking warfarin. Compared to young healthy volunteers, older inpatients had increased fibrin generation and decreased thrombin generation. The findings reflect the complex interaction between age, frailty, acute illness, and coagulation.

Increased thrombin generation in a mouse model of cancer cachexia is partially interleukin‐6 dependent

Background Cancer cachexia and cancer-associated thrombosis are potentially fatal outcomes of advanced cancer, which have not previously been mechanistically linked. The colon 26 (C26) carcinoma is a well-established mouse model of complications of advanced cancer cachexia, partially dependent on high levels of interleukin-6 (IL-6) produced by the tumor. Objectives To assess if cancer cachexia altered the coagulation state and if this was attributable to tumor IL-6 production. Methods In male BALB/c*DBA2 (F1 hybrid) mice with a C26 tumor we used modified calibrated automated thrombogram and fibrin generation (based on overall hemostatic potential) assays to assess the functional coagulation state, and also examined fibrinogen, erythrocyte sedimentation rate (ESR), platelet count, tissue factor pathway inhibitor (TFPI) and hepatic expression of coagulation factors by microarray. C26 mice were compared with non-cachectic NC26, pair-fed and sham control mice. IL-6 expression in C26 cells was knocked down by lentiviral shRNA constructs. Results C26 mice with significant weight loss and highly elevated IL-6 had elevated thrombin generation, fibrinogen, ESR, platelets and TFPI compared with all control groups. Fibrin generation was elevated compared with pair-fed and sham controls but not compared with NC26 tumor mice. Hepatic expression of coagulation factors and fibrinolytic inhibitors was increased. Silencing IL-6 in the tumor significantly, but incompletely, attenuated the increased thrombin generation, fibrinogen and TFPI. Conclusions Cachectic C26 tumor-bearing mice are in a hypercoagulable state, which is partly attributable to IL-6 release by the tumor. The findings support the importance of the coagulation state in cancer cachexia and the clinical utility of anti-inflammatory interventions.

The Overall Hemostatic Potential (OHP) Assay.

The principle of the overall hemostatic potential (OHP) assay is the generation of a fibrin time curve by optical density readings, which represent the balance between fibrin generation, triggered by thrombin or tissue factor, and fibrinolysis, triggered by tissue plasminogen activator (tPA). OHP measures the integrated effect of procoagulant, anticoagulant, and fibrinolytic factors, and OHP assay parameters provide a means of quantifying both the coagulation and fibrinolytic potential of platelet poor plasma. In particular, the OHP assay can be used to evaluate hypercoagulable states and abnormalities of fibrinolysis which are not well defined by assays which are routinely available in the coagulation laboratory. OHP is a technically simple assay, with potential for application in the routine laboratory at minimal cost.

Reaction of the hemocoagulation system to tissue hypoxia in patients with chronic obstructive pulmonary disease

Background. Nowadays little data related to the hemostatic system and fibrinolysis in patients with chronic obstructive pulmonary disease (COPD) are available. This is due to the lack of standardized methods for studying the hemostasis system, as well as to the lack of a single functional test that allows the evaluation of the complete fibrinogenesis cycle in whole blood. Aim. The aim of our study was to develop a functional test capable of analyzing the blood gas composition in the “point-of-care test” method for the evaluation of the hemostatic potential in patients with COPD, based on a standardized test stimulus, which is tissue hypoxia. The current level of clinical and laboratory diagnostics requires personification and research of the hemo-coagulation system in real time (point-of-care test), which allows low-frequency piezotromboelastography(NVTEG) to be performed. Materials and methods. NVTEG was chosen to estimate the state of the hemocoagulation system. Ten patients with COPD and 10 healthy volunteers were examined. Hypoxia was selected as a standardized test stimulus. Hypoxia conditions were caused by smoking one standard cigarette (composition: resin 10 mg/cig., nicotine 0,7 mg/cig., CO 10 mg/cig.). The degree of tissue hypoxia was assessed with the GASTAT-navi blood gas analyzer. Results. The study has shown that in response to the standard test stimulus, which is the tissue hypoxia caused by smoking of a standardized cigarette, two types of haemostatic potential reaction were detected both in patients with COPD and healthy volunteers. The first type of reaction – “hypercoagulation” – is characterized by the formation of chronometric and structural hypercoagulation at all stages of fibrinogenesis and increased coagulation activity by 25–30% compared with the response in healthy individuals. The second type of reaction – “hypocoagulation” – is characterized by the formation of chronometric and structural hypocoagulation, a decrease in coagulation activity by 25–30% compared with the response in healthy individuals. Conclusion. Test stimulus, which acts as tissue hypoxia, causes a uniform spectrum of changes in the blood gas composition and hemocoagulation system in both healthy volunteers and patients with COPD. The possibility of online assessment of all stages of fibrinogenesis makes it possible to stratify patients with COPD by type of reaction, which is certain to have an important diagnostic and prognostic value and in the future will allow a more personified approach for choosing the treatment tactics.

Possibility of application of low-frequency piezothromboelastografy method for the evaluation of haemostatic potential of blood in coronary bypass surgery on the background of long aspirinotherapy

Aim. To assess the hemostatic system on the aspirin therapy background using the VerifyNow Aspirin test, as well as examining platelet aggregation and low-frequency piezothromboelastography in patients with coronary artery disease during coronary artery bypass grafting. Materials and methods. 100 people with coronary artery disease who were taking aspirin (75–100 mg daily) for more than 1 year were examined. The study was performed in the perioperative period of coronary bypass surgery without aspirin withdrawal. Evaluation of hemostasis was performed by the VerifyNow Aspirin тest (USA), platelet aggregation (Нelena Laboratories AggRAMTM, Britain) and the low-frequency piezothromboelastography (ARP-01M “Mednord”, Russia). Results. Patients included in the study were sensitive to aspirin when under long-term administration of a drug: indicator test was VerifyNow ARU 496,9 ± 21,3)%, the platelet aggregation to ADP and adrenaline was reduced by 46% and 52%, respectively. In the early postoperative period platelet aggregation of ADP decreased by 73,2%, collagen – 75,9%, and adrenaline – 82,64% in comparison with the control group. Perioperative hemorrhagic complications in the study group were not observed. Reduction of platelet aggregation after aspirin therapy was accompanied by an increase in thrombin activity of the blood, which allows for evaluation of the method of low-frequency piezothromboelastography (LPTEG). In the early postoperative period, the results of LPTEG, thrombin potential, and anticoagulant and fibrinolytic activity of blood were partially normalized without reaching the level of the control group. Conclusion. For the evaluation of hemostasis under aspirin therapy it is advisable to apply the low-frequency piezothromboelastography, which in contrast to the VerifyNow test and traditional platelet aggregation, allows one to reveal a degree of impairment in thrombin blood activity and to conduct an integrative assessment on all aspects of hemostasis.

Evaluation of the hemostatic potential at coronary artery bypass surgery during long-term aspirin therapy

To estimate thrombin generation test parameters in patients with coronary heart disease during coronary artery bypass surgery under extracorporeal circulation after transfusion of donor platelet concentrates during long-term therapy with acetylsalicylic acid (ASA). A total of 148 patients with coronary heart disease who had undergone elective primary coronary artery bypass surgery under extracorporeal circulation during preserved therapy with ASA (75-100 mg/day) were examined. According to donor platelet concentrate transfusion, all the patients were divided into 2 groups: 1) 76 patients undergoing donor platelet transfusion and 2) 72 without this procedure. A control group consisted of 20 apparently healthy individuals. At the pre-, intra-, and early postoperative stages, the investigators evaluated the following thrombin generation test parameters: lag time (min); peak thrombin concentration (nM/l); time to peak (min); the area under the thrombin generation curve (nM), and thrombin generation rate (nM/min). During long-term ASA therapy, the patients were found to have an activated endogenous thrombin potential in the pre- and intraoperative periods, as evidenced by the high peak concentration of thrombin and the increased rate of its generation. At the same time, the time of prothrombinase complex activation and that of thrombin generation were longer than those in the control group. In the early postoperative period, the patients who had not been transfused with platelet concentrates with a further increase in the temporal parameters, showed a decreased hemostatic potential, reaching the control level, whereas donor platelet transfusion stimulated endogenous thrombin generation: the time to initiate clotting and that to reach the peak were shorter; in this case, the thrombin generation rate and concentrations increased, but the preoperative level was not reached. No perioperative (hemorrhagic or thrombotic ischemic) events were noted in the examined groups. The hemostatic potential was preserved in patients receiving long-term therapy for ASA. Taking into account laboratory and clinical findings, platelet concentrate transfusions are unnecessary for preventive purposes. The appropriateness of donor platelet transfusion should be strictly individually approached with regard to the laboratory parameters of the thrombin generation test, by minimizing the risk of perioperative ischemic and hemorrhagic events in each specific patient.

A New Classification of Mild/Moderate Hemophilia Α Based on a Multi-Dimensional FVIII(a) Functions Evaluated By a Plasma-Based Factor Xa Generation

In mild/moderate hemophilia A (MHA), the real hemostatic potential of their innate factor (F) VIII(a) may not be evaluated by FVIII activity (FVIII:C) obtained in a one-stage clotting assay. We often experience the discrepancies between clinical phenotype and FVIII:C in MHA patients. In order to elucidate the hemostatic potentials of the innate FVIII in MHA patients with a simple method, we here have established a plasma-based FXa generation assay (PB-XaG) to clarify the multi-dimensional functions of FVIII(a) in MHA determined by the interaction with the components of tenase complex consisting of FIXa, FX, and phospholipid (PL). XaG was evaluated in the patients’ plasmas and in the control plasma dilutions consisting of FVIII-deficient plasma and serial dilutions of recombinant FVIII (1-100 IU/dL) by a chromogenic substrate S-2765 together with human plasma-derived (pd)FIXa (0-20 nM), pdFX (0-200 nM), and PL vesicles (0-114 µM) initiated by thrombin (0.01 nM). The interaction between FVIIIa and each component of tenase complex was evaluated by Michaelis-Menten’s kinetic parameters, Vmax and apparent Km (Kmapp). We investigated the PB-XaG in the plasmas obtained from 17 MHA patients, carrying F8 mutations and FVIII:C as shown in the table. The PB-XaG showed that the group A, consisting of the case 1, 2, 3 and 4 with mutations at the residue(s) essential to formation of the inter-domain surface in the FVIII molecule, possessed ~1.8-7.1-fold increases in Kmapp for FIXa (10±0.9, 49±17, 31±6 and 9±3 nM, respectively) compared to FVIII:C-matched control (6.5±1.3 nM), whilst Kmapp for FX and PL in this group were of little difference from that in control, indicative of the reduced affinity for FIXa. The group B, including the case 5, 6, 7, 8 with mutations at the A2 or A3 domain remote from the PL-binding region on the C2 domain (residue 2303-2332) and the case 9 with the mutation at the PL-binding region, demonstrated ~1.7-4.8-fold increases in Kmapp for PL (4.3±2.4, 5.0±1.0, 6.5±1.3, 12±4.0 and 3.0±0.6 µM, respectively) compared to control (2.0±0.4 µM), whilst Kmapp for FIXa and FX in this group were similar to that in control, indicative of the reduced affinity to PL. Noteworthy, the apparent catalytic efficiencies defined as Vmax/Kmapp were 0.033 min-1 for the case 6 and 0.019 min-1 for the case 8 with mutations at the A3 domain, 0.3 or 0.1-fold lower than that for the case 9 (0.19 min-1) with mutation at the PL-binding region. The PB-XaG showed that the group C consisting of the case 10, 11, 12, 13, 14 and 15 demonstrated ~1.6-7.5-fold increases in Kmapp for FX (22±9, 23±9, 24±9, 10±4, 4.6±1.6 and 5.8±3.0 nM, respectively) compared to control (4.5±0.8 nM), whilst Kmapp for FIXa and PL in this group were similar to that in control, indicative of the reduced affinity to FX. Interestingly, the case 16 classified in the group C’ showed enhancing affinity with FX represented by a half decrease of Kmapp for FX compared to FVIII:C-matched control (2.0±0.9 and 4.8±1.5 nM, respectively). By contrast, the case 17 with mutation at the thrombin cleavage site categorized in the group D demonstrated any little XaG initiated by thrombin for all axes of FIXa, FX and PL. Taken together, the function of FVIII(a) in MHA could be evaluated multi-dimensionally and clearly classified into the some types of interaction with FIXa, FX, PL and thrombin by the PB-XaG. Our plasma-based XaG assay would be a useful tool for functional analyses of FVIII(a) to clarify the mechanism(s) of hemostatic heterogeneity in mild/moderate HA. [Display omitted] DisclosuresNogami:Chugai Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Sysmex Corporation: Patents & Royalties, Research Funding. Shima:F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Sysmex Corporation: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding.