COVID-19 patient plasma demonstrates resistance to tPA-induced fibrinolysis as measured by thromboelastography.

Patients critically ill with COVID-19 are at risk for thrombotic events despite prophylactic anticoagulation. Impaired fibrinolysis has been proposed as an underlying mechanism. Our objective was to determine if fibrinolysis stimulated by tissue plasminogen activator (tPA) differed between COVID patients and controls. Plasma from 14 COVID patients on prophylactic heparin therapy was obtained and compared with heparinized plasma from 14 different healthy donors to act as controls. Kaolin activated thromboelastography with heparinase was utilized to obtain baseline measurements and then repeated with the addition of 4 nM tPA. Baseline fibrinogen levels were higher in COVID plasma as measured by maximum clot amplitude (43.6 ± 6.9 mm vs. 23.2 ± 5.5 mm, p < 0.0001) and Clauss assay (595 ± 135 mg/dL vs. 278 ± 44 mg/dL, p < 0.0001). With the addition of tPA, fibrinolysis at 30 min after MA (LY30%) was lower (37.9 ± 16.5% vs. 58.9 ± 18.3%, p = 0.0035) and time to 50% lysis was longer (48.8 ± 16.3 vs. 30.5 ± 15.4 min, p = 0.0053) in the COVID-19 samples. Clotting times and rate of fibrin polymerization ('R' or 'α' parameters) were largely the same in both groups. Clot from COVID patients contains a higher fibrin content compared to standard controls and shows resistance to fibrinolysis induced by tPA. These findings suggest the clinical efficacy of thrombolytics may be reduced in COVID-19 patients.

Controlled Multifactorial Coagulopathy: Effects of Dilution, Hypothermia, and Acidosis on Thrombin Generation In Vitro.

BACKGROUND: Coagulopathy and hemostatic abnormalities remain a challenge in patients following trauma and major surgery. Coagulopathy in this setting has a multifactorial nature due to tissue injury, hemodilution, hypothermia, and acidosis, the severity of which may vary. In this study, we combined computational kinetic modeling and in vitro experimentation to investigate the effects of multifactorial coagulopathy on thrombin, the central enzyme in the coagulation system. METHODS: We measured thrombin generation in platelet-poor plasma from 10 healthy volunteers using the calibrated automated thrombogram assay (CAT). We considered 3 temperature levels (31°C, 34°C, and 37°C), 3 pH levels (6.9, 7.1, and 7.4), and 3 degrees of dilution with normal saline (no dilution, 3-fold dilution, and 5-fold dilution). We measured thrombin-generation time courses for all possible combinations of these conditions. For each combination, we analyzed 2 scenarios: without and with (15 nM) supplementation of thrombomodulin, a key natural regulator of thrombin generation. For each measured thrombin time course, we recorded 5 quantitative parameters and analyzed them using multivariable regression. Moreover, for multiple combinations of coagulopathic conditions, we performed routine coagulation tests: prothrombin time (PT) and activated partial thromboplastin time (aPTT). We compared the experimental results with simulations using a newly developed version of our computational kinetic model of blood coagulation. RESULTS: Regression analysis allowed us to identify trends in our data (P < 10). In both model simulations and experiments, dilution progressively reduced the peak of thrombin generation. However, we did not experimentally detect the model-predicted delay in the onset of thrombin generation. In accord with the model predictions, hypothermia delayed the onset of thrombin generation; it also increased the thrombin peak time (up to 1.30-fold). Moreover, as predicted by the kinetic model, the experiments showed that hypothermia increased the area under the thrombin curve (up to 1.97-fold); it also increased the height of the thrombin peak (up to 1.48-fold). Progressive acidosis reduced the velocity index by up to 24%; acidosis-induced changes in other thrombin generation parameters were much smaller or none. Acidosis increased PT by 14% but did not influence aPTT. In contrast, dilution markedly prolonged both PT and aPTT. In our experiments, thrombomodulin affected thrombin-generation parameters mainly in undiluted plasma. CONCLUSIONS: Dilution with normal saline reduced the amount of generated thrombin, whereas hypothermia increased it and delayed the time of thrombin accumulation. In contrast, acidosis in vitro had little effect on thrombin generation.

Effects of blood storage age on immune, coagulation, and nitric oxide parameters in transfused patients undergoing cardiac surgery.

BACKGROUND: Retrospective studies suggested that storage age of RBCs is associated with inflammation and thromboembolism. The Red Cell Storage Duration Study (RECESS) trial randomized subjects undergoing complex cardiac surgery to receive RBCs stored for shorter versus longer periods, and no difference was seen in the primary outcome of change in multiple organ dysfunction score. STUDY DESIGN AND METHODS: In the current study, 90 subjects from the RECESS trial were studied intensively using a range of hemostasis, immunologic, and nitric oxide parameters. Samples were collected before transfusion and on Days 2, 6, 28, and 180 after transfusion. RESULTS: Of 71 parameters tested, only 4 showed a significant difference after transfusion between study arms: CD8+ T-cell interferon-γ secretion and the concentration of extracellular vesicles bearing the B-cell marker CD19 were higher, and plasma endothelial growth factor levels were lower in recipients of fresh versus aged RBCs. Plasma interleukin-6 was higher at Day 2 and lower at Days 6 and 28 in recipients of fresh versus aged RBCs. Multiple parameters showed significant modulation after surgery and transfusion. Most analytes that changed after surgery did not differ based on transfusion status. Several extracellular vesicle markers, including two associated with platelets (CD41a and CD62P), decreased in transfused patients more than in those who underwent surgery without transfusion. CONCLUSIONS: Transfusion of fresh versus aged RBCs does not result in substantial changes in hemostasis, immune, or nitric oxide parameters. It is possible that transfusion modulates the level of platelet-derived extracellular vesicles, which will require study of patients randomly assigned to receipt of transfusion to define.

A Step Toward Balance: Thrombin Generation Improvement via Procoagulant Factor and Antithrombin Supplementation.

BACKGROUND: The use of prothrombin complex concentrates in trauma- and surgery-induced coagulopathy is complicated by the possibility of thromboembolic events. To explore the effects of these agents on thrombin generation (TG), we investigated combinations of coagulation factors equivalent to 3- and 4-factor prothrombin complex concentrates with and without added antithrombin (AT), as well as recombinant factor VIIa (rFVIIa), in a dilutional model. These data were then used to develop a computational model to test whether such a model could predict the TG profiles of these agents used to treat dilutional coagulopathy. METHODS: We measured TG in plasma collected from 10 healthy volunteers using Calibrated Automated Thrombogram. TG measurements were performed in undiluted plasma, 3-fold saline-diluted plasma, and diluted plasma supplemented with the following factors: rFVIIa (group rFVIIa); factors (F)II, FIX, FX, and AT (group "combination of coagulation factors" [CCF]-AT); or FII, FVII, FIX, and FX (group CCF-FVII). We extended an existing computational model of TG to include additional reactions that impact the Calibrated Automated Thrombogram readout. We developed and applied a computational strategy to train the model using only a subset of the obtained TG data and used the remaining data for model validation. RESULTS: rFVIIa decreased lag time and the time to thrombin peak generation beyond their predilution levels (P < 0.001) but did not restore normal thrombin peak height (P < 0.001). CCF-FVII supplementation decreased lag time (P = 0.034) and thrombin peak time (P < 0.001) and increased both peak height (P < 0.001) and endogenous thrombin potential (P = 0.055) beyond their predilution levels. CCF-AT supplementation in diluted plasma resulted in an improvement in TG without causing the exaggerated effects of rFVIIa and CCF-FVII supplementation. The differences between the effects of CCF-AT and supplementation with rFVIIa and CCF-FVII were significant for lag time (P < 0.001 and P = 0.005, respectively), time to thrombin peak (P < 0.001 and P = 0.004, respectively), velocity index (P < 0.001 and P = 0.019, respectively), thrombin peak height (P < 0.001 for both comparisons), and endogenous thrombin potential (P = 0.034 and P = 0.019, respectively). The computational model generated subject-specific predictions and identified typical patterns of TG improvement. CONCLUSIONS: In this study of the effects of hemodilution, CCF-AT supplementation improved the dilution-impaired plasma TG potential in a more balanced way than either rFVIIa alone or CCF-FVII supplementation. Predictive computational modeling can guide plasma dilution/supplementation experiments.

Optimizing Thrombin Generation with 4-Factor Prothrombin Complex Concentrates in Neonatal Plasma After Cardiopulmonary Bypass.

BACKGROUND: Bleeding is a serious complication after pediatric cardiopulmonary bypass (CPB) that is associated with an increase in perioperative morbidity and mortality. Four-factor prothrombin complex concentrates (4F-PCCs) have been used off-label to supplement transfusion protocols for bleeding after CPB in adults; however, data on their use in neonates are limited. In this study, we hypothesized that 4F-PCCs administered ex vivo to neonatal plasma after CPB will increase thrombin generation. METHODS: Fifteen neonates undergoing complex cardiac repairs requiring CPB were enrolled in this prospective study. Arterial blood was obtained after anesthesia induction but before CPB (baseline), after CPB following heparin reversal, and after our standardized transfusion of a quarter of a platelet apheresis unit (approximately 20 mL·kg) and 3 units of cryoprecipitate. Kcentra (CSL Behring), a 4F-PCC with nonactivated factor VII (FVII), and factor 8 inhibitor bypassing activity (FEIBA; Baxter Healthcare Corporation), a 4F-PCC with activated FVII, were added ex vivo to plasma obtained after CPB to yield concentrations of 0.1 and 0.3 IU·mL. Calibrated automated thrombography was used to determine thrombin generation for each sample. RESULTS: The addition of Kcentra to plasma obtained after CPB resulted in a dose-dependent increase in the median (99% confidence interval) peak amount of thrombin generation (42.0 [28.7-50.7] nM for Kcentra 0.1 IU·mL and 113.9 [99.0-142.1] nM for Kcentra 0.3 IU·mL). The rate of thrombin generation was also increased (15.4 [6.5-24.6] nM·min for Kcentra 0.1 IU·mL and 48.6 [29.9-66.6] nM·min for Kcentra 0.3 IU·mL). The same was true for FEIBA (increase in peak: 39.8 [27.5-49.2] nM for FEIBA 0.1 IU·mL and 104.6 [92.7-124.4] nM for FEIBA 0.3 IU·mL; increase in rate: 17.4 [7.4-28.8] nM·min FEIBA 0.1 IU·mL and 50.5 [26.7- 63.1] nM·min FEIBA 0.3 IU·mL). In the posttransfusion samples, there was a significant increase with Kcentra in the median (99% confidence interval) peak amount (41.1 [21.0-59.7] nM for Kcentra 0.1 IU·mL and 126.8 [106.6- 137.9] nM for Kcentra 0.3 IU·mL) and rate (18.1 [-6.2 to 29.2] nM·min for Kcentra 0.1 IU·mL and 53.2 [28.2-83.1] nM·min for Kcentra 0.3 IU·mL) of thrombin generation. Again, the results were similar for FEIBA (increase in peak: 43.0 [36.4-56.7] nM for FEIBA 0.1 IU·mL and 109.2 [90.3-136.1] nM for FEIBA 0.3 IU·mL; increase in rate: 25.0 [9.1-32.6] nM·min for FEIBA 0.1 IU·mL and 59.7 [38.5-68.7] nM·min for FEIBA 0.3 IU·mL). However, FEIBA produced in a greater median reduction in lag time of thrombin generation versus Kcentra in samples obtained after CPB (P = 0.003 and P = 0.0002 for FEIBA versus Kcentra at 0.1 and 0.3 IU·mL, respectively) and in samples obtained after transfusion (P < 0.0001 for FEIBA versus Kcentra at 0.1 and 0.3 IU·mL). CONCLUSIONS: After CPB, thrombin generation in neonatal plasma was augmented by the addition of 4F-PCCs. The peak amount and rate of thrombin generation were enhanced in all conditions, whereas the lag time was shortened more with FEIBA. Our findings suggest that the use of 4F-PCCs containing activated FVII may be an effective adjunct to the initial transfusion of platelets and cryoprecipitate to augment coagulation and control bleeding in neonates after CPB.

Computational simulation and comparison of prothrombin complex concentrate dosing schemes for warfarin reversal in cardiac surgery.

BACKGROUND: Prothrombin complex concentrate (PCC) is increasingly used for acute warfarin reversal. We hypothesized that computational modeling of thrombin generation (TG) could be used to optimize the timing and dose of PCC during hemodilution induced by cardiopulmonary bypass (CPB). METHODS: Thrombin generation patterns were modeled in anticoagulated patients (n = 59) using a published computational model. Four dosing schemes were evaluated including single full dose (median, 41.2 IU/kg) of PCC before or after CPB, ½-dose before and after CPB, or 1/3-dose before CPB plus 2/3-dose after CPB. Hemodilution was modeled as 40 or 60 % dilution of factors from baseline. The lag time (s) of TG, and peak thrombin level (nM) were evaluated. RESULTS: Prolonged lag time, and reduced peak TG were due to low vitamin K-dependent (VKD) factors, and pre-CPB PCC dose-dependently restored TG to near-normal or normal range. After 40 % dilution, TG parameters were similar among 4 regimens at the end of therapy. The recovery of VKD factors was less when PCC was given before CPB after 60 % dilution, but TG parameters were considered hemostatically effective (>200 nM) with any regimen. Withholding the full dose of PCC until post-CPB resulted in severely depressed TG peak (median, 47 nM) after 60 % dilution, and some supra-normal TG peaks after treatment. CONCLUSIONS: Pre-CPB administration of full or divided doses of PCC prevents extremely low TG peak during surgery, and maintains hemostatic TG peaks in both 40 and 60 % hemodilution models. Although PCC's hemostatic activity appears to be highest using the full dose after CPB, hypercoagulability may develop in some cases.

Transfusion and hematologic variables after fibrinogen or platelet transfusion in valve replacement surgery: preliminary data of purified lyophilized human fibrinogen concentrate versus conventional transfusion.

BACKGROUND: Platelet (PLT) and plasma transfusion remain the mainstay hemostatic therapy for perioperative bleeding. Several studies have indicated that acquired fibrinogen (FIB) deficiency can be the primary cause of bleeding after cardiac surgery. The aim of this study was to compare hematologic and transfusion profiles between the first-line FIB replacement and PLT transfusion in post-cardiac surgical bleeding. STUDY DESIGN AND METHODS: In this prospective, randomized, open-label study, 20 adult patients who underwent valve replacement or repair and fulfilled preset visual bleeding scale were randomized to 4 g of FIB or 1 unit of apheresis PLTs. Primary endpoints included hemostatic condition in the surgical field and 24-hour hemostatic product usage. Hematologic data, clinical outcome, and safety data were collected up to the 28th day postoperative visit. RESULTS: In patients who received the first-line FIB concentrate (n = 10), the visual bleeding scale improved after intervention, and the incidence of PLT transfusion and total plasma donor exposure were lower compared to the PLT group (n = 10). Postintervention FIB level was statistically higher (209 mg/dL vs. 165 mg/dL) in the FIB group than in the PLT group, but PLT count and prothrombin were lower. There were no statistical differences in the postoperative blood loss and red blood cell transfusion between two groups. CONCLUSIONS: Our preliminary data indicate that the primary FIB replacement may potentially reduce the incidence of PLT transfusion and the number of donor exposures. Plasma FIB level of 200 mg/dL is attainable with a single dose of 4 g, and this level seems to mitigate bleeding despite moderately decreased thrombin generation.

Detection of cellular traction forces via the force-triggered Cas12a-mediated catalytic cleavage of a fluorogenic reporter strand.

Molecular forces generated by cell receptors are infrequent and transient, and hence difficult to detect. Here we report an assay that leverages the CRISPR-associated protein 12a (Cas12a) to amplify the detection of cellular traction forces generated by as few as 50 adherent cells. The assay involves the immobilization of a DNA duplex modified with a ligand specific for a cell receptor. Traction forces of tens of piconewtons trigger the dehybridization of the duplex, exposing a cryptic Cas12-activating strand that sets off the indiscriminate Cas12-mediated cleavage of a fluorogenic reporter strand. We used the assay to perform hundreds of force measurements using human platelets from a single blood draw to extract individualized dose-response curves and half-maximal inhibitory concentrations for a panel of antiplatelet drugs. For seven patients who had undergone cardiopulmonary bypass, platelet dysfunction strongly correlated with the need for platelet transfusion to limit bleeding. The Cas12a-mediated detection of cellular traction forces may be used to assess cell state, and to screen for genes, cell-adhesion ligands, drugs or metabolites that modulate cell mechanics.

A comparative evaluation of rotation thromboelastometry and standard coagulation tests in hemodilution-induced coagulation changes after cardiac surgery.

BACKGROUND: Coagulopathy after cardiopulmonary bypass (CPB) is caused by multiple perturbations in cellular and humoral elements of coagulation. A timely and comprehensive method to evaluate hemostasis would be helpful in the management of bleeding patients after CPB. The assessment of whole blood coagulation using rotation thromboelastometry (ROTEM) was compared to coagulation tests routinely performed during cardiac surgery. STUDY DESIGN AND METHODS: Blood was obtained from 26 patients undergoing CPB surgery at baseline, at 60 minutes on CPB, at the end of CPB, and on admission to intensive care unit. ROTEM tests (extrinsically activated [EXTEM], intrinsically activated [INTEM], specific clot formation [FIBTEM]), prothrombin time, activated partial thromboplastin time, platelet (PLT) count, fibrinogen, prothrombin level, antithrombin level, and thrombin generation (TG) measurement were performed. RESULTS: We observed strong correlations between FIBTEM-amplitude at 10 minutes (A10) and fibrinogen level (r=0.87; p<0.001) and between EXTEM/ INTEM-A10 variables and PLT count (r=0.72 and 0.67, respectively; p<0.001). Receiver operating characteristic analysis demonstrated that EXTEM-A10 and INTEM-A10 are predictive of thrombocytopenia below 80×10(9)/L (area under the curve [AUC], 0.83 and 0.82, respectively), and FIBTEM-A10 was highly predictive of fibrinogen level below 200 mg/dL (AUC, 0.96). There were only weak correlations found between TG peak and clot formation time of EXTEM or INTEM (r=0.30 and 0.29, respectively; p<0.05). CONCLUSION: ROTEM variables demonstrated clinically relevant correlations with PLT counts and fibrinogen levels. In particular, decreasing levels of fibrinogen can be quickly determined (<15-20 min) using FIBTEM.

Fibrinogen and hemostasis: a primary hemostatic target for the management of acquired bleeding.

Fibrinogen plays several key roles in the maintenance of hemostasis. Its cleavage by thrombin and subsequent polymerization to form fibrin strands provides the structural network required for effective clot formation. During cases of acute blood loss, attempts to maintain circulating volume and tissue perfusion often involve the infusion of crystalloids, colloids, and red blood cells. Intravascular volume resuscitation, although vital, frequently results in dilution of the remaining clotting factors and onset of dilutional coagulopathy. In such cases, fibrinogen is the first coagulation factor to decrease to critically low levels. There currently is a lack of awareness among physicians regarding the significance of fibrinogen during acute bleeding and, at many centers, fibrinogen is not monitored routinely during treatment. We reviewed current studies that demonstrate the importance of considering fibrinogen replacement during the treatment of acquired bleeding across clinical settings. If depleted, the supplementation of fibrinogen is key for the rescue and maintenance of hemostatic function; however, the threshold at which such intervention should be triggered is currently poorly defined. Although traditionally performed via administration of fresh frozen plasma or cryoprecipitate, the use of lyophilized fibrinogen (concentrate) is becoming more prevalent in some countries. Recent reports relating to the efficacy of fibrinogen concentrate suggest that it is a viable alternative to traditional hemostatic approaches, which should be considered. The prospective study of fibrinogen supplementation in acquired bleeding is needed to accurately assess the range of clinical settings in which this management strategy is appropriate, the most effective method of supplementation and a comprehensive safety profile of fibrinogen concentrate used for such an approach.

The impact of hematocrit on fibrin clot formation assessed by rotational thromboelastometry.

BACKGROUND: Rotational thromboelastometry (ROTEM®)-based FIBTEM is used perioperatively to assess the extent of fibrin polymerization in whole blood. In FIBTEM, cytochalasin D eliminates the contribution of platelets to whole blood clotting, but changing levels in fibrin(ogen) and erythrocytes may differently affect clot formation. Because dynamic changes of hematocrit are not reflected in plasma fibrinogen measurements, we hypothesized that the lack of erythrocytes in isolated plasma measurements would affect the relationship between the Clauss method and whole blood-based FIBTEM during cardiac surgery. Therefore, in the current study we investigated the influence of perioperative hematocrit changes on FIBTEM and fibrinogen measurements. METHODS: Blood samples were collected from 6 consenting healthy volunteers. FIBTEM tests were run before and after serial in vitro dilutions of whole blood with saline or autologous plasma (5:1, 2:1, and 1:1 v/v). We then evaluated the relationship between FIBTEM-maximal clot firmness (MCF) and the Clauss fibrinogen method in relation to hematocrit values before and after cardiac surgery. Pearson correlation coefficients were determined between laboratory test results and ROTEM variables. RESULTS: Upon in vitro hematocrit reduction, FIBTEM-MCF was progressively decreased depending on the extent of saline dilution, but it was increased by 31% after 1:1 volume replacement with autologous plasma (P < 0.05). In samples from cardiac patients (150 measurements in 50 patients), the overall correlation coefficient between FIBTEM-MCF and plasma fibrinogen was 0.80 (P < 0.001). In hemodiluted blood samples (during surgery or at intensive care unit), FIBTEM-MCF 10 mm corresponded to plasma fibrinogen levels of 200 mg/dL. In the subgroup analysis (n = 50 each), according to hematocrit levels (<25%, ≥25% to 30%, ≥30%), plasma fibrinogen levels of 200 mg/dL corresponded to 11 mm, 10 mm, and 8 mm of FIBTEM-MCF, respectively. The correlation between FIBTEM-MCF and plasma fibrinogen was higher at lower hematocrit (<25%) than at higher hematocrit (>30%) (r = 0.88 and 0.67, respectively). CONCLUSIONS: Perioperative changes in hematocrit affect the correlation between plasma fibrinogen levels and FIBTEM-MCF values. The higher correlation between FIBTEM-MCF and plasma fibrinogen with lower hematocrit (<25%) indicates that FIBTEM is a practical method to determine the need for fibrinogen replacement in bleeding patients who typically develop perioperative anemia.

The effects of MDCO-2010, a serine protease inhibitor, on activated clotting time in blood obtained from volunteers and cardiac surgical patients.

BACKGROUND: The activated clotting time (ACT) is widely used for monitoring heparin anticoagulation during cardiac surgery. Celite-based ACT values are prolonged when aprotinin is administered. MDCO-2010, a novel serine protease inhibitor, is currently being evaluated as a possible alternative to aprotinin. Therefore, we evaluated the in vitro effects of this novel agent on ACT values using 3 different point-of-care instruments with kaolin or celite as an activator. METHODS: The study was performed in 2 parts. In the first part, blood samples were obtained from 15 healthy volunteers. Samples were pipetted into small Eppendorf tubes and 2 concentrations of the MDCO-2010 (100 and 500 nM, final concentration) alone or with heparin (1.2 or 2.4 U/mL) were added. ACTs were measured using Helena (celite), Hemochron (kaolin), and Medtronic (kaolin) devices. In the second part of the study, blood samples were obtained intraoperatively, at 5 time points, from 15 patients undergoing cardiopulmonary bypass. MDCO-2010 at a final concentration of 100 or 500 nM was added and ACT testing was performed as before. Additional coagulation tests included prothrombin time, activated partial thromboplastin time, fibrinogen, antithrombin, prothrombin, and anti-Xa levels. RESULTS: Addition of MDCO-2010 concentration-dependently prolonged ACTs in volunteers' and patients' blood samples regardless of the ACT activator or device used. In volunteer samples (no heparin) and in patient samples (baseline and intensive care unit) percent changes in ACTs due to MDCO-2010 were on average 3.1 ± 1.8 times higher (95% confidence interval 2.6-3.6; P < 0.001) for the celite-based Helena device compared with either Hemochron or Medtronic devices. CONCLUSION: MDCO-2010 causes less ACT prolongation with kaolin than with celite activation.

COVID-19 patient fibrinogen produces dense clots with altered polymerization kinetics, partially explained by increased sialic acid.

BACKGROUND: Thrombogenicity is a known complication of COVID-19, resulting from SARS-CoV-2 infection, with significant effects on morbidity and mortality. OBJECTIVE: We aimed to better understand the effects of COVID-19 on fibrinogen and the resulting effects on clot structure, formation, and degradation. METHODS: Fibrinogen isolated from COVID-19 patients and uninfected subjects was used to form uniformly concentrated clots (2 mg/ml), which were characterized using confocal microscopy, scanning electron microscopy, atomic force microscopy, and endogenous and exogenous fibrinolysis assays. Neuraminidase digestion and subsequent NANA assay were used to quantify sialic acid residue presence; clots made from the desialylated fibrinogen were then assayed similarly to the original fibrinogen clots. RESULTS: Clots made from purified fibrinogen from COVID-19 patients were shown to be significantly stiffer and denser than clots made using fibrinogen from noninfected subjects. Endogenous and exogenous fibrinolysis assays demonstrated that clot polymerization and degradation dynamics were different for purified fibrinogen from COVID-19 patients compared with fibrinogen from noninfected subjects. Quantification of sialic acid residues via the NANA assay demonstrated that SARS-CoV-2-positive fibrinogen samples contained significantly more sialic acid. Desialylation via neuraminidase digestion resolved differences in clot density. Desialylation did not normalize differences in polymerization, but did affect rate of exogenous fibrinolysis. DISCUSSION: These differences noted in purified SARS-CoV-2-positive clots demonstrate that structural differences in fibrinogen, and not just differences in gross fibrinogen concentration, contribute to clinical differences in thrombotic features associated with COVID-19. These structural differences are at least in part mediated by differential sialylation.

Identification of coagulation factor IX variants with enhanced activity through ancestral sequence reconstruction.

Orthologous proteins contain sequence disparity guided by natural selection. In certain cases, species-specific protein functionality predicts pharmacological enhancement, such as greater specific activity or stability. However, immunological barriers generally preclude use of nonhuman proteins as therapeutics, and difficulty exists in the identification of individual sequence determinants among the overall sequence disparity. Ancestral sequence reconstruction (ASR) represents a platform for the prediction and resurrection of ancient gene and protein sequences. Recently, we demonstrated that ASR can be used as a platform to facilitate the identification of therapeutic protein variants with enhanced properties. Specifically, we identified coagulation factor VIII (FVIII) variants with improved specific activity, biosynthesis, stability, and resistance to anti-human FVIII antibody-based inhibition. In the current study, we resurrected a panel of ancient mammalian coagulation factor IX (FIX) variants with the goal of identifying improved pharmaceutical candidates. One variant (An96) demonstrated 12-fold greater FIX activity production than human FIX. Addition of the R338L Padua substitution further increased An96 activity, suggesting independent but additive mechanisms. after adeno-associated virus 2 (AAV2)/8-FIX gene therapy, 10-fold greater plasma FIX activity was observed in hemophilia B mice administered AAV2/8-An96-Padua as compared with AAV2/8-human FIX-Padua. Furthermore, phenotypic correction conferred by the ancestral variant was confirmed using a saphenous vein bleeding challenge and thromboelastography. Collectively, these findings validate the ASR drug discovery platform as well as identify an ancient FIX candidate for pharmaceutical development.

The anticoagulant effect of protamine sulfate is attenuated in the presence of platelets or elevated factor VIII concentrations.

BACKGROUND: Protamine sulfate is the antidote for heparin, but in excess it exerts weak anticoagulation. METHODS: We evaluated the effects of increasing protamine concentrations (0 to 24 microg/mL) on prothrombin time and diluted Russell's viper venom time measurements on thrombin generation in platelet-poor and platelet-rich plasma after activation by tissue factor or actin, and on thromboelastometry in platelet-poor plasma and whole blood from 6 healthy volunteers. The reversibility of excess protamine (24 microg/mL) by recombinant factor VIIa or factor VIII/von Willebrand factor concentrate was also tested. RESULTS: Protamine prolonged prothrombin time and Russell's viper venom time, concentration dependently. Protamine also increased lag time and decreased peak of thrombin generation in platelet-poor plasma after tissue factor and actin activation. In platelet-rich plasma with platelets at 50 to 200 x 10(3)/microL, protamine (24 microg/mL) prolonged the lag time, but had no effect on peak thrombin generation. The addition of factor VIII/von Willebrand factor (1.5-3.0 U/mL) to platelet-poor plasma with protamine (24 microg/mL) decreased lag time and increased peak thrombin generation with actin activation. A therapeutic concentration of recombinant factor VIIa (60 nM) only affected the lag time of thrombin generation triggered with actin. In agreement, protamine increased coagulation time evaluated by thromboelastometry significantly more in platelet-poor plasma than in whole blood. CONCLUSIONS: We demonstrated that protamine affects the propagation of thrombin generation, which is partially reversed by platelets or increased factor VIII/von Willebrand factor concentrations. The present data suggest that excess protamine might potentially increase bleeding in the case of severe thrombocytopenia or low factor VIII.

In vitro comparative study of hemostatic components in warfarin-treated and fibrinogen-deficient plasma.

OBJECTIVE: The authors hypothesized that various hemostatic products may differently affect viscoelastic clot formation depending on their respective procoagulant activity and fibrinogen content. DESIGN: In vitro coagulopathy modeling using warfarin-treated plasma (international normalized ratio, 2.8-3.8) and fibrinogen-deficient plasma evaluated by rotational thromboelastometry (ROTEM; Pentapharm, Munich, Germany). SETTING: A university laboratory. INTERVENTION: Different volumes of cryoprecipitate, fresh frozen plasma (FFP), fibrinogen concentrate, and platelet concentrate were mixed with each abnormal plasma to simulate the in vivo transfusions of 250 mL to 1,000 mL. Three thromboelastometric variables that reflect the rate and extent of clot growth were measured: (1) coagulation time (CT), (2) angle, and (3) maximal clot firmness (MCF). MEASUREMENTS AND MAIN RESULTS: In warfarin-treated plasma, the addition of FFP, cryoprecipitate, and platelets led to a dose-dependent improvement of CT and angle, whereas MCF increased with cryoprecipitate or platelets only. The addition of fibrinogen concentrate improved MCF and angle but not CT. In fibrinogen-deficient plasma, the addition of cryoprecipitate, platelets, and fibrinogen concentrate led to a dose-dependent improvement of ROTEM variables, whereas the addition of FFP resulted in significantly longer CT and lower MCF values compared with other hemostatic products. The addition of platelets in the presence of cytochalasin D (a platelet inhibitor) resulted in improvements of ROTEM variables that were similar to when FFP was added to warfarin-treated and fibrinogen-deficient plasma. CONCLUSIONS: Cryoprecipitate supports clot formation on ROTEM more efficiently than FFP because of the high fibrinogen content. Improved ROTEM variables after platelet addition are presumably caused by increased interaction among thrombin-activated platelets and fibrinogen.

In-vitro evaluation of anti-factor IXa aptamer on thrombin generation, clotting time, and viscoelastometry.

The REG1 system consists of factor IXa inhibitor, RB006, an aptamer-based anticoagulant and its antidote, RB007. The optimal use of RB006 can be facilitated by understanding its effect on the formation of thrombin and fibrin, and other standard tests of coagulation. Blood from consented volunteers was drawn into 3.2% citrate (9:1 v/v) and either used immediately or centrifuged to obtain platelet-poor plasma. Increasing concentrations of aptamer (6-24 microg/ml) alone or in combination with heparin (0.1 U/ml) or lepirudin (0.2 microg/ml) were added to blood and plasma samples. Activated clotting times (ACT+, low range-ACT), thrombelastometry (ROTEM) or thrombelastography (TEG) were performed in recalcified whole blood samples. Thrombin generation, prothrombin time (PT) and activated partial thromboplastin time (aPTT) were performed in plasma samples. To some samples the antidote RB007 was added to neutralise the anticoagulation activity of RB006. In all experiment the ratio of RB006 to RB007 was kept 1:2. RB006 dose-dependently prolonged aPTT and low range-ACT, but, as expected, had no effect on PT. RB006 prolonged the lag time and decreased the peak of Actin-triggered thrombin generation. Thrombin-activated TEG demonstrated that RB006 decreases the rate of clot formation. These effects were potentiated when RB006 was combined with heparin or lepirudin. In all experiments RB007 reversed the effects of RB006 back to baseline. In conclusion, RB006 inhibits thrombin generation and clot formation in a concentration-dependent manner. It is feasible to monitor RB006 and its reversal with RB007 using aPTT, low range-ACT, and thrombin-activated TEG.

Platelet function, but not thrombin generation, is impaired in acute normovolemic hemodilution (ANH) blood.

STUDY OBJECTIVE: We investigated the coagulation changes that might occur in acute normovolemic hemodilution (ANH) blood over several hours during cardiac surgery requiring cardiopulmonary bypass. DESIGN: This study was a prospective observational study. SETTING: This study took place at a university teaching hospital. PATIENTS: This study included 26 patients, either ASA 3 or 4 and without known coagulation disorders, undergoing cardiac surgery. Patients were included if the use of cardiopulmonary bypass was expected to reach 2.5 h. INTERVENTIONS: ANH blood was collected into CPDA-1 collection bags before systemic heparinization. Samples were taken directly from the bags at time of collection and reinfusion to assess changes in platelet and thrombin generation parameters. MEASUREMENTS: Whole blood from citrated tubes was used immediately for rotational thromboelastometry and platelet aggregometry analyses. Thrombin generation was assessed using calibrated automated thrombography with platelet poor plasma. MAIN RESULTS: Despite no significant change in platelet count over the ANH storage period, there was significant degradation in platelet function as measured by thrombin receptor activating peptide stimulation on Mulltiplate™ analysis and maximum clot formation on ROTEM™ EXTEM. Notably, there was no change in the ability to generate thrombin. CONCLUSIONS: Little data exists regarding the quality of coagulation factors in autologous blood. Our study confirms ANH collection results in decreased platelet aggregation with TRAP stimulation; however, this is not appreciated with ADP stimulation. Thrombin generation capacity remains preserved.