Co-Administration of Factor XIII with Hemostatic Agents in Hemophilia Promotes Clot Stability and Composition

Abstract Hemophilia A results from the absence, deficiency, or inhibition of factor VIII. Bleeding is treated with use of two broad classes of hemostatic agents; replacement factors (recombinant or plasma-derived human FVIII [FVIII] or factor IX) or bypassing agents (recombinant activated factor VIIa [rFVIIa], plasma-derived anti-inhibitor coagulant complex [FEIBA], or recombinant porcine FVIII [rpFVIII]). Despite treatment with hemostatic agents, some hemophilia patients experience refractory bleeding. Factor XIII-A2B2 (FXIII) is a thrombin-activated protransglutaminase. Activated FXIII (FXIIIa) crosslinks fibrin to stabilize clots and increase red blood cell retention during clot contraction, and therefore, clot weight; these functions have been specifically associated with fibrin alpha-chain crosslinking and production of high molecular weight (HMW) crosslinked fibrin species. Although pilot studies have indicated that in hemophilia co-administration of FXIII with hemostatic agents improves hemostasis, the biochemical mechanisms evoked during co-administration have yet to be elucidated. We hypothesize that co-administration of FXIII with hemostatic agents enhances hemostasis in hemophilia by accelerating FXIII activation, increasing fibrin crosslinking, and improving the composition of contracted clots. We first analyzed in vitrotissue factor-activated coagulation in FVIII-deficient plasmas (n=5) with or without buffer (normal saline with 0.75% bovine serum albumin), plasma-derived FXIII (2 U/mL), and/or recombinant human FVIII (1 U/mL). Reactions were performed at 37°C and quenched with urea/EDTA at varying time points. Clot lysates were separated on 10% Tris-glycine gels, blotted, probed for FXIII(a) and fibrin(ogen), and analyzed by densitometry. Compared to buffer, FXIII, or FVIII-treated hemophilic plasma, FVIII+FXIII co-treatment increased the formation rate and level of FXIIIa. Furthermore, compared to buffer or FXIII-treated hemophilic plasma, both FVIII-treatment and FVIII+FXIII co-treatment increased the formation rate and level of crosslinked fibrin species (gamma chain and HMW species). Notably, compared to FVIII-treatment (alone), FVIII+FXIII co-treatment increased the formation rate and level of HMW crosslinked fibrin species. Next, we analyzed clot formation in whole blood obtained from untreated FVIII-deficient (n=2) or FVIII-inhibitor (n=6) patients. Citrated whole blood was pre-incubated at 37°C for 30 minutes with or without buffer, FXIII (2 U/mL), rFVIIa (25 nM), FEIBA (1 U/mL), or rpFVIII (1 U/mL), alone and in combination with FXIII, and clot formation was triggered with tissue factor and recalcification. In thrombelastography assays, compared to buffer or FXIII-treated hemophilic whole blood, rFVIIa, FEIBA, and rpFVIII treatments alone or with FXIII co-treatment shortened the time to clot formation (clot time [R]). Compared to buffer, FXIII, FEIBA or rpFVIII-treated hemophilic whole blood, rFVIIa-treatment (alone) and co-treatment with rFVIIa+FXIII or FEIBA+FXIIII increased clot stability (area under the elastic curve). In whole blood clot contraction assays, compared to buffer, FXIII, rFVIIa, FEIBA, or rpFVIII-treated hemophilic whole blood, co-treatment with rFVIIa+FXIII, FEIBA+FXIII, or rpFVIII+FXIII significantly increased clot weight. Our data show that in hemophilic plasmas, co-administration of FXIII with hemostatic agents accelerates FXIIIa activation, resulting in accelerated and increased HMW species formation. Furthermore, in hemophilic whole blood, co-administration of FXIII with hemostatic agents improved clot biophysical characteristics and increased clot weight. Collectively, these data suggest co-administration of FXIII with conventional hemostatic agents may promote clot stability and improve the composition of contracted clots by enhancing fibrin alpha-chain crosslinking. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Recombinant Factor VIIa Combined with Plasma Factor XIII Stabilize Clot Formation and Increase Clot Stability in Whole Blood from Patients with Severe Hemophilia A.

Blood ◽

10.1182/blood.v108.11.1039.1039 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1039-1039 ◽

Cited By ~ 1

Author(s):

Benny Sorensen ◽

Rasmus Rojkjaer ◽

Jorgen Ingerslev

Keyword(s):

Whole Blood ◽

Factor Xiii ◽

Hemophilia A ◽

Recombinant Factor Viia ◽

Factor Viia ◽

Ex Vivo ◽

Clot Formation ◽

Severe Hemophilia ◽

Clot Strength ◽

Clot Stability

Abstract Patients with severe hemophlia A and inhibitors suffer from significantly compromised clot formation as well as reduced clot stability. Recombinant factor VIIa (rFVIIa - NovoSeven®, Novo Nordisk, Bagsvaerd, Denmark) has proven safe and efficacious for securing haemostasis in hemophilia patients with inhibitors. Recently, it was proposed that the reduced thrombin generation in severe haemophilia hinders sufficient activation of factor XIII and thereby result in insufficient covalent lateralization of fibrin (Blood2005; 106: 11, Abstract 321). The present study aimed at exploring the effect of rFVIIa and rFVIIa + plasma-derived FXIII (Haematological Technologies Inc) on whole blood clot (WB) formation and WB clot stability in severe hemophilia A. In total, 14 patients with a verified FVIII:C < 0.01 IU/ml were enrolled. Ex vivo studies were performed with rFVIIa (2 μg/ml), rFVIIa+FXIII (2+10 μg/ml), and a buffer control. Dynamic WB coagulation profiles describing initiation (clotting time=CT[sec]), propagation (maximum velocity=MaxVel [mm*100/sec]) and clot strength (maximum clot firmness=MCF[mm*100]) were recorded using thrombelastography and activation with a minute amount of tissue factor (TF, Innovin, final dilution 1:50000). WB clot stability was evaluated using a reaction mixture containing TF and tPA (1nM), followed by evaluation of the MCF and the total area under the elasticity curve after 120 min analysis time (AUEC[mm*100*sec]). Data are presented as mean and Wilcoxon statistical results. In the absence of tPA, Both rFVIIa+FXIII and rFVIIa significantly shortened the CT (Buffer=1424, rFVIIa+FXIII=739 (p=0.010), rFVIIa=881, (p=0.0005)) and accelerated WB MaxVel (Buffer=3.8, rFVIIa+FXIII=10.5 (p=0.0001), rFVIIa=9.2, (p=0.0002)). The standard deviation (SD) of the CT was significant lower in WB spiked with rFVIIa+FXIII than rFVIIa (Buffer SD=697, rFVIIa+FXIII SD=289 vs rFVIIa SD=655, p=0.007). In the absence of tPA, rFVIIa+FXIII increased the MCF significantly more than rFVIIa (Buffer=4441, rFVIIa+FXIII=6414 vs rFVIIa=5943, p=0.04) and the SD of the MCF was significant lower in WB spiked with rFVIIa+FXIII than rFVIIa (Buffer SD=2174, rFVIIa+FXIII SD=331 vs rFVIIa SD=948, p=0.0006). In the presence of tPA, rFVIIa+FXIII induced higher clot strength and stability than rFVIIa alone (MCF: Buffer=1313, rFVIIa+FXIII=3295 vs rFVIIa=3023, p=0.10 (N.S.); AUEC: Buffer=3.8*106, rFVIIa+FXIII=12.8*106 vs rFVIIa=10.2*106, p=0.0269). In conclusion, both rFVIIa (2μg/mL) and FXIII (10 μg/ml) added to rFVIIa (2 μg/ml), significantly increased WB clot formation and stability in this ex vivo evaluation of the clotting potential of WB from patients with severe hemophilia A.

Download Full-text

The Haemocompatibility of Biomaterials In Vitro: Investigations on the Mechanism of the Whole-blood Clot Formation Test

Alternatives to Laboratory Animals ◽

10.1177/026119299202000305 ◽

1992 ◽

Vol 20 (3) ◽

pp. 390-395 ◽

Cited By ~ 1

Author(s):

Thomas Groth ◽

Katrin Derdau ◽

Frank Strietzel ◽

Frank Foerster ◽

Hartmut Wolf

Keyword(s):

Whole Blood ◽

Blood Clotting ◽

Blood Clot ◽

Formation Rate ◽

Clot Formation ◽

Coagulation Cascade ◽

Contact Activation ◽

Human Fibrinogen ◽

Static Conditions

Twenty years ago Imai & Nose introduced a whole-blood clotting test for the estimation of haemocompatibility of biomaterials in vitro In our paper a modification of this assay is described and the mechanism of clot formation further elucidated. It was found that neither the inhibition of platelet function nor the removal of platelets from blood significantly changed the clot formation rate on glass and polyvinyl chloride in comparison to the rate tor whole blood. Scanning electron microscopy demonstrated that platelets were not involved in clot formation near the blood/biomaterial interface. Thus, it was concluded that the system of contact activation of the coagulation cascade dominates during clot formation under static conditions. The latter conclusion was supported by the fact that preadsorption of human serum albumin or human fibrinogen onto the glass plates used, decreased the clot formation rate in the same manner.

Download Full-text

In Vitro Inhibition of Factor XIII Retards Clot Formation, Reduces Clot Firmness, and Increases Fibrinolytic Effects in Whole Blood

Anesthesia & Analgesia ◽

10.1213/ane.0b013e3181b5a263 ◽

2009 ◽

Vol 109 (4) ◽

pp. 1023-1028 ◽

Cited By ~ 30

Author(s):

Csilla Jámbor ◽

Viviane Reul ◽

Thomas W. Schnider ◽

Priska Degiacomi ◽

Hubert Metzner ◽

...

Keyword(s):

Whole Blood ◽

Factor Xiii ◽

Clot Formation ◽

In Vitro Inhibition

Download Full-text

The Effects of Thrombocytopenia on Clotting Profile in Whole Blood As Determined By Thromboelastography When Anticoagulant Is Present at Therapeutic or Prophylactic Concentration

Blood ◽

10.1182/blood.v124.21.1550.1550 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1550-1550

Author(s):

Jason Chung ◽

Ivan Stevic ◽

Jorell Gantioque ◽

Anthony K.C. Chan ◽

Howard H.W. Chan

Keyword(s):

Anticoagulant Therapy ◽

Whole Blood ◽

Statistical Difference ◽

Conflicts Of Interest ◽

Maximum Amplitude ◽

Clot Formation ◽

Bleeding Tendency ◽

Therapeutic Concentration ◽

Significant Difference

Abstract Background: Anticoagulant therapy for the treatment of venous thromboembolism in patients with concomitant thrombocytopenia has been based on anecdotal evidence. The platelet (PLT) threshold at which anticoagulant therapy should be withheld is still controversial. A PLT count of 50 × 109/L was recommended to be the threshold in the past, but newer reviews have lowered the threshold to 30 × 109/L. We previously used thromboelastography (TEG) to study clotting in plasma reconstituted with autologous PLT. Since red cells also play a significant role in hemostasis and coagulation, we hereby developed a TEG model with whole blood (WB) in order to better mimic in vivo conditions to evaluate the clot formation in thrombocytopenic blood. Objective: Using TEG to monitor clotting in whole blood samples containing unfractionated heparin (UFH) or dalteparin, we evaluated the differences in clotting profile when PLT in the samples were reduced to thrombocytopenic range. Methods: Whole blood was collected from healthy volunteers in syringes containing citrate phosphate dextrose adenine (CDPA-1, pH=5.5) and 30 μg/L corn trypsin inhibitor. Magnetic CD 61 antibody chromatography was used to deplete PLT in the blood to a count of ≤ 15 × 109/L. Platelet-depleted whole blood (PDWB) was then mixed with untouched blood from the same donor to obtain the predefined PLT counts. Clotting was initiated in the TEG cups with 10 mM CaCl2 and tissue factor (TF) in the presence of either UFH (0.3 U/mL or 0.1 U/mL) or dalteparin (1 IU/mL or 0.3 IU/mL). Due to the mechanistic differences between UFH and dalteparin, we optimized the amount of TF to maximize the sensitivity of TEG assay for individual anticoagulants; thus, 2.25 pM and 2.05 pM were used for UFH and dalteparin experiments, respectively. However, the same amount of TF was used to evaluate the clotting with the same anticoagulant at both therapeutic and prophylactic concentrations. Clotting was monitored using a Haemoscope TEG at 37 ºC for a maximum of 3 hr or until maximum amplitude (MA) had been achieved. Three parameters of clotting profile including R, MA and area under the curve within the first 15 min of clotting (AUC15) were used for further analysis. A p-value < 0.05 was considered statistically significant. Results: All3 parameters showed significant compromise of clotting when PLT decreased from 150 × 109/L to < 15 × 109/L in the presence of UFH or dalteparin at therapeutic range. When these anticoagulants were reduced to prophylactic concentration, the clotting was also significantly moderated, but to a lesser extent, comparing samples with PLT at 150 × 109/L and those with PLT < 15 × 109/L. These are in accordance with the bleeding tendency in vivo. At 30 × 109/L, the newer recommended PLT threshold at which anticoagulant should be withheld in thrombocytopenic patients, the clotting parameters did not show any significant difference as compared to those at the traditional threshold of 50 × 109/L when UFH and dalteparin were at therapeutic concentrations. Similarly, when UFH was reduced to a prophylactic concentration, we detected no significant difference in the clotting profile between 50 × 109 PLT/L and 30 × 109 PLT/L. In contrast, in samples with dalteparin at a prophylactic concentration, MA was significantly lower at 30 × 109 PLT/L when compared with that at 50 × 109 PLT/L although R and AUC15 had no statistical difference. Additionally, samples of PDWB containing either anticoagulant at prophylactic concentration had better clot formation than those samples of 50 × 109 PLT/L containing UFH or dalteparin at therapeutic concentration. Conclusion: The TEG profile of WB clotting in this in vitro model simulates bleeding tendency observed clinically. In the presence of UFH or dalteparin at therapeutic concentration, there was no statistical difference in the TEG parameters comparing thrombocytopenic blood with 50 × 109 PLT/L and 30 × 109 PLT/L, supporting the latter as the new threshold to hold anticoagulant in thrombocytopenic patients. In addition, instead of holding all anticoagulants in severe thrombocytopenic patients with PLT < 30 × 109/L, administering UFH or dalteparin at prophylactic doses may offer a safe alternative, as both imped clotting in TEG even less than those at therapeutic concentration with thrombocytopenic blood at 50 × 109 PLT/L. Fig 1. TEG profile of clots with UFH Fig 1. TEG profile of clots with UFH Fig 2. TEG profile of clots with dalteparin Fig 2. TEG profile of clots with dalteparin Disclosures No relevant conflicts of interest to declare.

Download Full-text

In vitro factor XIII supplementation increases clot firmness in Rotation Thromboelastometry (ROTEM®)

Thrombosis and Haemostasis ◽

10.1160/th09-12-0858 ◽

2010 ◽

Vol 104 (08) ◽

pp. 385-391 ◽

Cited By ~ 73

Author(s):

Lars Asmis ◽

Burkhardt Seifert ◽

Donat Spahn ◽

Oliver Theusinger ◽

Werner Baulig

Keyword(s):

Factor Xiii ◽

Clot Formation ◽

Intensive Care Patients ◽

Essential Parameter ◽

Α Angle ◽

The Impact ◽

Clot Formation Time ◽

Clot Stability ◽

Maximum Clot Firmness

SummaryFactor XIII (F XIII) is an essential parameter for final clot stability. The purpose of this study was to determine the impact of the addition of factor (F)XIII on clot stability as assessed by Rotation Thromboelastometry (ROTEM®). In 90 intensive care patients ROTEM® measurements were performed after in vitro addition of F XIII 0.32 IU, 0.63 IU, 1.25 IU and compared to diluent controls (DC; aqua injectabile) resulting in approximate F XIII concentrations of 150, 300 and 600%. Baseline measurements without any additions were also performed. The following ROTEM® parameters were measured in FIBTEM and EXTEM tests: clotting time (CT), clot formation time (CFT), maximum clot firmness (MCF), maximum lysis (ML), maximum clot elasticity (MCE) and α-angle (αA). Additionally, laboratory values for FXIII, fibrinogen (FBG), platelets and haematocrit were contemporaneously determined. In the perioperative patient population mean FBG concentration was elevated at 5.2 g/l and mean FXIII concentration was low at 62%. The addition of FXIII led to a FBG concentration-dependent increase in MCF both in FIBTEM and EXTEM. Mean increases in MCF (FXIII vs. DC) of approximately 7 mm and 6 mm were observed in FIBTEM and EXTEM, respectively. F XIII addition also led to decreased CFT, increased αA, and reduced ML in FIBTEM and EXTEM. In vitro supplementation of FXIII to supraphysiologic levels increases maximum clot firmness, accelerates clot formation and increases clot stability in EXTEM and FIBTEM as assayed by ROTEM® in perioperative patients with high fibrinogen and low FXIII levels.

Download Full-text

Small-Volume Noncontact Assessment of Blood Coagulation Via Acoustic Tweezing Coagulometry

Blood ◽

10.1182/blood-2021-153819 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3178-3178

Author(s):

Nithya Kasireddy ◽

Elizabeth M Cummins ◽

Huy Q Pham ◽

Amina Rafique ◽

Damir B Khismatullin

Keyword(s):

Blood Plasma ◽

Blood Coagulation ◽

Whole Blood ◽

American Heart Association ◽

American Heart ◽

Formation Rate ◽

Heart Association ◽

Sample Volume ◽

Clot Formation ◽

Fviii Inhibitor

Abstract Introduction: Blood coagulation analysis is routinely performed to assess bleeding and thrombotic risks in surgical and critical care patients as well as in patients with diseases that cause coagulation abnormalities (e.g., hemophilia, thrombophilia and sickle cell disease). Majority of coagulation assays are based on photo-optical measurement of coagulation onset in blood plasma such as prothrombin time (PT), international normalized ratio (INR), and activated partial thromboplastin time (aPTT) and viscoelastic measurement of coagulating whole blood, often referred to as "global coagulation analysis", mostly done by thromboelastography (TEG, ROTEM) but they require large sample volume (> 0.5ml) requiring venipuncture, have poor standardization, and are unreliable tools to predict bleeding/thrombotic risk. Acoustic tweezing coagulometry (ATC) is an innovative noncontact drop-of-blood coagulation analysis technique that can perform both photo-optical and viscoelastic coagulation analysis with a sample volume as low as 4 μl to provide a comprehensive set of clinically relevant coagulation parameters such as blood viscosity, elasticity, reaction time, clotting rate, maximum clot stiffness, fibrin formation rate and cross-linking kinetics helpful for diagnosis and prediction of bleeding and thrombotic risks. ATC is particularly valuable for the pediatric patients as it enables safe and reliable point of care coagulation assessment with minimal sample volume. Materials and Methods: In this project, we demonstrate the feasibility of ATC for coagulation analysis by validation and standardization of the technique using whole blood collected from healthy adult volunteers and commercially purchased blood plasma. Further, we present the ability of ATC to assess bleeding risk in commercial blood plasma with coagulation FVIII deficiency with and without inhibitors, as well as whole blood collected from pediatric Hemophilia A patients without inhibitors. The time dependent changes in elasticity (elastic tweezograph, Figure 1A) and viscosity (viscous tweezograph, Figure 1B) of coagulating blood plasma or whole blood sample are used to extract the following coagulation parameters: clot initiation time (CIT), clotting rate (CR), clotting time (CT), time to firm clot formation (TFCF), and maximum clot stiffness (MCS) from elastic tweezograph; reaction time (RT), fibrin formation rate (FFR), and maximum fibrin level (MFL) from viscous tweezograph. Results and Discussion: Figure 1C shows the elastic tweezograph and figure 1D shows the viscous tweezograph of the healthy plasma, plasma with coagulation FVIII deficieny and plasma with inhibitors for coagulation FVIII activated via the intrinsic pathway of coagulation. The tweezographs suggest that the clot initiation is faster in healthy plasma compared to the FVIII deficient plasma and FVIII inhibitor plasma. The clotting rate is highest for healthy plasma followed by the FVIII deficient plasma and is the lowest for the plasma with FVIII inhibitors suggesting a delayed clot formation in the deficient and inhibitor groups. They all reach a similar final clot stiffness, but the time to firm clot formation is least in healthy plasma as expected and increases in the FVIII deficient group and further increases in the FVIII inhibitor group. Conclusions: Acoustic tweezing coagulometry can successfully measure the viscosity, elasticity and coagulation of whole blood and blood plasma with only a drop of the sample. This technique can successfully assess the bleeding risks in pediatric and adult patients with Hemophilia. Acknowledgements: This study has been supported by American Heart Association pre doctoral fellowship 20PRE35210991, U.S. National Science Foundation grant 1438537, American Heart Association Grant-in-Aid 13GRNT17200013, and Tulane University intramural grants. The acoustic tweezing technology is protected by pending patents PCT/US14/55559, PCT/US2018/014879 and PCT/US21/15336. Figure 1 Figure 1. Disclosures Kasireddy: Levisonics Inc.: Current Employment. Rafique: Pfizer Inc.: Consultancy; CSL Behring: Consultancy; HEMA Biologics: Consultancy. Khismatullin: Levisonics Inc.: Current equity holder in publicly-traded company; Levisonics Inc.: Patents & Royalties: PCT/US14/55559 (pending); Levisonics Inc.: Patents & Royalties: PCT/US2018/014879 (issued) ; Levisonics Inc.: Patents & Royalties: PCT/US21/15336 (pending)..

Download Full-text

Fibrinogen: A Procoagulant and An Anticoagulant

Blood ◽

10.1182/blood.v118.21.384.384 ◽

2011 ◽

Vol 118 (21) ◽

pp. 384-384 ◽

Cited By ~ 1

Author(s):

Catherine J Rea ◽

Benny Sorensen ◽

Jørgen Ingerslev ◽

Peter Laursen

Keyword(s):

Tissue Factor ◽

Whole Blood ◽

Thrombin Generation ◽

Plasma Concentrations ◽

Lag Time ◽

Clot Formation ◽

Fibrinogen Concentration ◽

Clotting Time ◽

Dose Dependent ◽

Clot Stability

Abstract Abstract 384 Fibrinogen: A Procoagulant and an Anticoagulant Introduction: Bleeding occurs secondary to acquired fibrinogen deficiency but the effect of high fibrinogen is more controversial. Correlation between raised fibrinogen levels and venous or arterial thrombosis has been recorded. However, fibrinogen increases as an acute phase response and may be an innocent biomarker, detected at elevated levels in individuals with concomitant disease. Recent animal studies provide evidence that high fibrinogen does not trigger thrombosis per se, but enhances thrombotic occlusion of vessels following tissue injury. Aims: This study aims to investigate the effect of elevated levels of fibrinogen on thrombin generation and clot resistance to accelerated fibrinolysis. We hypothesised that fibrinogen promotes clot stability following a high tissue factor stimulus (TF), but will act as an anticoagulant following low TF stimulus. Method: Normal human plasma was spiked with fibrinogen to achieve final plasma concentrations of 2.7, 3.2, 3.7, 4.7, 5.7, 6.7, 8 and 11.7g/l. Coagulation was initiated with TF at variable dilutions (1:20000, 1: 500) plus calcium. To assess clot stability the same assay was performed with simultaneous addition of tissue plasminogen activator (t-Pa 0.75nmolar). Clot formation and lysis was recorded via light absorbance (FLUOstar Omega). Clot stability was also measured by whole blood thromboelastometry; citrate and CTI stabilized whole blood was drawn from a healthy individual and spiked with fibrinogen (calculated plasma concentrations: 3.2, 4, 3.9, 5.5, 9.9, 16 g/l). Coagulation was triggered with TF (1: 50000 or 1:500), calcium and of t-Pa (2nmolar). The area under elasticity curve (AUEC) at 90mins was the primary endpoint. Thrombin generation in plasma was performed in plasma following addition of fibrinogen using fluorogenic substrate and calcium (FluCä, Thrombinoscope BV, The Netherlands). Results:Plasma clot formation assay: Dose dependent shortening of clot time and time to peak turbidity were seen with increasing fibrinogen following a high TF stimulus (TF 1:500) (figure 1- panel A). Conversely, following a low TF stimulus increasing fibrinogen caused a lengthening of the clotting time. Plasma and whole blood lysis assays: With high TF stimulus, fibrinogen produced a dose-dependent increase in clot stability measures (AUC/AUEC) in both plasma and whole blood assays (figure 1-panel B). Following a low TF stimulus increases in the fibrinogen concentration resulted in suppressed clot stability. Thrombin generation: A decrease in total thrombin generation was seen with increasing fibrinogen (Figure 2) at both high and low TF levels. With high TF there was no alteration in lag-time, but with low TF stimulus the lag-time progressively lengthened as fibrinogen concentration increased. Discussion: Fibrinogen acts as a pro-coagulant by promoting clot formation and supports clot stability following a high TF stimulus. However, following a low TF stimulus elevated fibrinogen becomes an anticoagulant as demonstrated by prolonging clotting time and decreases clot stability in both plasma and whole blood. In conclusion, our data suggest that elevated fibrinogen per se is not thrombogenic. However, following a significant trauma resulting in a high tissue factor stimulus and high thrombin generation, fibrinogen acts predominantly as a pro-coagulant enhancing clot formation and supporting clot stability. This may protect against bleeding or contribute to pathological thrombotic events. In contrast, following a minor trauma prompting a minimal tissue factor stimulus, fibrinogen predominantly acts as an anticoagulant and may protect against thrombosis. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Platelets from Fresh and Cold-Stored Whole Blood Contribute to Clot Formation after Transfusion in Rats with Acute Traumatic Coagulopathy

Blood ◽

10.1182/blood.v128.22.1457.1457 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1457-1457

Author(s):

Xiaowu Wu ◽

Robbie Montgomery ◽

Bin Liu ◽

Jeffrey D Keesee ◽

Avi Benov ◽

...

Keyword(s):

Whole Blood ◽

Bleeding Time ◽

Conflicts Of Interest ◽

Tissue Injury ◽

Clot Formation ◽

Trauma Patients ◽

Acute Traumatic Coagulopathy ◽

Liver Sample ◽

Arterial Blood ◽

Traumatic Coagulopathy

Abstract Background: The hemostatic and platelet functions of fresh whole blood (FWB) are well preserved when stored at 4°C for 14 days (human) or 7 days (rats). Resuscitation of either FWB or whole blood stored for 7-days at 4°C (SWB) in rats with acute traumatic coagulopathy (ATC) induced by polytrauma and hemorrhage (T/H), shows identical correction of hemodynamic and metabolic function, as well as the equivalent impact on platelet aggregation in vitro. Platelet hemostatic function is influenced by platelet number, aggregation, and platelet-leukocyte interactions. Therefore, the objective of this study is to identify the in-vivo fate and activity of platelets from FWB and SWB, and determine whether transfused platelets contribute to clot formation in tissue injury after transfusion. Method: Isoflurane anesthetized Sprague-Dawley rats underwent polytrauma, followed by 40% hemorrhage. The rats were resuscitated (20% of bled volume) 1hr after trauma using either FWB or SWB (n=5 each) collected from green fluorescence (GFP) donor rats. SWB was kept in mini-blood-bag and refrigerated at 40C for 7 days, and warmed up to 370C prior to transfusion. Sham rats had no trauma, but underwent 20% blood replacement by FWB or SWB (n=4 each). Immediately after transfusion, a liver incision was made, followed by the measurement of kidney bleeding time (both standardized incisions was made by a Surgicutt adult template device). The incisional liver sample was taken 10min after injury for immunohistochemistry stained for CD61 (platelets) and GFP. Blood samples taken from donor rats (FWB and SWB) and recipient rats at baseline, immediately before resuscitation, and 0min and 10min after transfusion were analyzed by flow cytometry using CD42d, CD45, and CD62P antibodies for defining platelets, leukocytes and activated platelets respectively. Lactadherin was used to detect phosphatidylserine (PS). Platelet-leukocyte aggregates were defined as CD42d+/CD45+. The transfused and native platelet or leukocyte was differentiated by GFP signal. Results: GFP signal intensity was equally expressed in platelets derived from either FWB or SWB. Storage at 4°C led to a significant increase in number of platelets expressing CD62P (76±2% vs. 11±4%) and PS (8.6±0.7% vs. 1.1±0.4%) as compared to FWB. SWB also showed a significant rise in intensity, but not percentage, of platelet-leukocyte aggregates (45781±6235 vs. 10978±2345 MFI). After transfusion, recipient rats showed a significant elevation in the percentage of GFP+ platelets after transfusion of FWB as compared to SWB (Sham:13.0±1.0% vs. 4.9±0.4%; T/H: 17.0±0.8% vs. 6.8±0.%). Similarly, GFP+ leukocytes aggregates from FWB were three times higher than SWB (Sham: 6.15±0.5% vs. 1.9±0.5%; T/H: 4.4±0.4% vs. 1.2±0.2%). There was greater percentage of GFP+ platelet-leukocyte aggregates in both T/H and sham rats transfused with SWB than FWB (Sham: 11.7±3.3% vs. 33.8±2.3%; T/H: 9.8±1.6% vs. 47.2±5.1%). Transfusion with SWB led to a significant increase in percentage of activated GFP+ platelets in T/H rats as compared to FWB (30.0±2.7% vs. 2.5±0.5%). However, the activity of native platelets was not significantly different between SWB and FWB in T/H rats after transfusion (3.9±1.2% vs. 3.7±0.3%). The kidney bleeding time was not significantly different in T/H rats receiving FWB and SWB (131±4 vs.127±7 seconds) under equivalent mean arterial blood pressure (82±7 vs 85±9 mmHg). The clot that formed at the site of liver incision was identified by platelet aggregates stained by the CD61 antibody. Using co-localization of CD61 and GFP, we found that the platelets from both FWB and SWB equivalently incorporated into the clot at incisional site. Conclusion: The platelets from SWB are higher in platelet activation state, clearance rate and platelet-leukocyte aggregates than FWB after transfusion in both sham rats and rats with polytrauma and hemorrhage. However, platelets from both fresh and cold-stored whole blood contribute to hemostasis of tissue injury after transfusion. This study suggests that cold-stored whole blood is an alternative resource to treat trauma patients for restoration of hemostatic function. Future study is necessary to optimize the storage of whole blood to prolong the platelet survival rate and optimize hemostatic function. This project was funded by US Army Medical Research and Material Command. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Mechanism Underlying a Role for Factor XIII (FXIII) Polymorphism in Sickle Cell Disease-Associated Priapism

Blood ◽

10.1182/blood-2018-99-116946 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2361-2361

Author(s):

Marilyn J. Telen ◽

Milena Batchvarova ◽

Joan D Beckman ◽

Martha Delahunty ◽

Karen L Soldano ◽

...

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Whole Blood ◽

Research Funding ◽

Factor Xiii ◽

Cell Disease ◽

Risk Genotype ◽

Transglutaminase Activity ◽

Clot Contraction ◽

Fxiii Activity

Abstract Background: The pathophysiology of priapism in sickle cell disease (SCD) is poorly understood. While blood stasis is essential to tumescence, most research in SCD-associated priapism to date has focused on the potential role of abnormal signaling pathways. We have previously observed that a coding sequence single nucleotide polymorphism (rs5988) of the transglutaminase factor XIII gene (FXIII) is strongly associated with SCD priapism, with an odds ratio of 2.52 [C.I. 1.27 -5.03] for the risk genotype (G/G, expressing only FXIII E652) vs the most common non-risk genotype (G/C, expressing both FXIII E652 and FXIII Q652). We therefore explored the effect of the rs5988 polymorphism on various aspects of FXIII function. Methods: Recombinant FXIII (rFXIII) E652 and rFXIII Q652 were expressed by 293 kidney cells and isolated from serum-free tissue culture supernatant. Before use in assays, activation of rFXIII by thrombin was confirmed by generating activated rFXIII (rFXIIIa) with thrombin (10 U/ml), followed SDS-PAGE, western blotting for FXIII, and densitometry for quantitation. Whole blood samples and plasmas were obtained from previously genotyped subjects with SCD under an IRB-approved protocol. Plasma FXIII and rFXIII transglutaminase activity was measured by the ability to catalyze 5-(biotinamido)pentylamine incorporation into a suitable substrate. Plasma FXIII antigen was assayed by ELISA. Clot contraction was measured after tissue factor-initiated clotting of recalcified whole blood. Clot resistance to lysis was measured after exposure to tissue plasminogen activator (tPA). Results: Transglutaminase activity of each rFXIII was measured using fibrinogen and fibronectin as substrates. At 20 minutes, rFXIIIa E652 showed 1.44-fold more transglutaminase (crosslinking) activity toward fibrin(ogen) than rFXIIIa Q652 (p=0.027), and a nonsignificant trend toward more activity (1.32-fold, p=0.079) toward fibronectin. Kinetic assays also showed that rFXIII E652 had significantly greater activity toward both matrices (p=0.006 and 0.012, respectively), suggesting the risk genotype (homozygosity for the G allele) enhances fibrin(ogen) and/or fibronectin crosslinking. FXIII activity in the plasma of 18 genotyped SCD patients (3 CC, 7 GC, 8 GG) demonstrated a consistent, but not significant, trend toward increased FXIII activity with increasing presence of G alleles (80.01% CC, 98.90% CG, and 107.20% GG). Although results were not adjusted for genotype at other loci reported to affect FXIII expression, there was no significant difference in FXIII antigen among genotypes. Compared to contracted whole blood clots from patients with only one or no risk alleles, clots from SCD patients with two copies of the risk allele (GG, expressing only FXIII E652) did not differ in either RBC retention within clots or clot mass (weight). Moreover, inhibition of transglutaminase activity with T101 significantly increased RBC release and decreased clot weight to a similar degree in contracted clots from patients with either the GC or GG genotype. In clots formed from FXIII-deficient plasma supplemented with rFXIIIa E652 or rFXIIIa Q652 and ABO-compatible donor RBCs, clots containing rFXIIIa E652 were 14% more resistant to lysis than clots containing rFXIIIa Q652 (p=0.016). Parallel studies with SCD patient plasma samples also showed that clots containing only FXIII E652 were more resistant to lysis than clots containing both FXIII E652 and FXIII Q652(p=0.0001). Conclusions: These data suggest the FXIII rs5988 polymorphism does not alter protein expression or clot contraction but may regulate clot stability via slightly increased transglutaminase activity and enhanced resistance to lysis. These effects may predispose patients to formation of microclots during tumescence, thus impairing blood egress and increasing risk of priapism. Further studies should be conducted to determine if anticoagulation or fibrinolytic treatments are viable preventative or treatment strategies for SCD patients with the risk FXIIIGG genotype and recurrent priapism. Disclosures Telen: Pfizer, Inc.: Consultancy, Research Funding. Wolberg:GlaxoSmithKline: Employment; Novo Nordisk: Research Funding.

Download Full-text

Beyond Prothrombin Time and Activated Partial Thromboplastin Time: Clot Formation Rate and Clot Structure Index As Novel Parameters for Coagulation Test Running Head: Novel Parameters of Coagulation Test

Blood ◽

10.1182/blood.v128.22.4950.4950 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4950-4950

Author(s):

Jaewoo Song ◽

Yoon Jihoon ◽

Lee Minyong ◽

Yoon Tae Hyun

Keyword(s):

Disease Severity ◽

Conflicts Of Interest ◽

Formation Rate ◽

Clot Formation ◽

Fibrinogen Concentration ◽

Curve Shape ◽

Coagulation Test ◽

Waveform Data ◽

Structure Index ◽

Clot Structure

Abstract BACKGROUND: Coagulation analyzers based on turbidimetry provide clot waveform data. Clotting times are defined only a point on this curve and have limitations to represent the whole characteristics of curve. We investigated to represent the whole curve shape by additional parameters and explored its clinical utility. METHODS: We applied the Gompertz growth model to the waveform and the two-dimensional curve shape can be expressed with novel parameters. The model was evaluated in terms of fit, applicability, and reproducibility. To understand how the properties of parameters affected by various conditions, we controlled fibrinogen concentration, clot structure, and factor activity. For clinical application of these parameters, a reference interval was established, and the distribution was studied in different populations (normal, lupus anticoagulant-positive, and hemophilia). Finally, they were examined for use as indicators of disease severity in hemophilia and direct oral anticoagulant monitoring. RESULTS: The model was near-perfectly fitted to the waveform, mostly applicable, and reproducible. Two parameters of the model, κ and α, determined the shape of waveform; κ was linked to the fibrinogen content and clot structure, and α reflected clotting activity and influenced on κ inversely. The clot formation rate (CFR) and clot structure index (CSI), defined using κ and α, provided additive clinical information for hemostatic function evaluation, disease severity estimation, and anticoagulant monitoring beyond the clotting times. CONCLUSIONS: The clotting curve model enabled to utilize the overall shape of clot waveform. CFR and CSI which derived from the model can be used as additional informative parameters of coagulation test. Disclosures No relevant conflicts of interest to declare.

Download Full-text