scholarly journals Pharmacokinetics and Pharmacodynamics of Recombinant FIX Variants in the FIX Knockout Mouse Tail Clip Bleeding Model

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3180-3180
Author(s):  
Sabine Pestel ◽  
Hendrik Peil ◽  
Steffi Knoll Machado ◽  
Philipp Claar ◽  
Elmar Raquet ◽  
...  
Keyword(s):  
Blood Loss ◽  
Plasma Levels ◽  
Half Life ◽  
Bleeding Time ◽  
Isotonic Saline ◽  
Publicly Traded ◽  
Total Blood ◽  
Time Points ◽  
Hemostatic Activity ◽  
Tail Clip

Abstract Introduction: The recessive X-linked bleeding disorder Hemophilia B is caused by a mutation in the coagulation factor (F) IX gene leading to partial or total loss of its function. Preventive treatment with replacement long-acting FIX is an attractive option for patients to reduce administration frequency and prevent bleeding. New recombinant FIX therapeutics like the albumin-fused FIX (rFIX-FP) or the Fc-fused FIX (rFIX-Fc) enable longer half-life in circulation and thus less frequent administration, as compared to non-fused FIX (rFIX). Studies in FIX knockout (KO) mice were conducted to characterize the effect of the modifications on the pharmacokinetic (PK) and pharmacodynamic (PD) properties of the different recombinant FIX products. Methods: Pharmacokinetics: Recombinant FIXs were administered intravenously at doses of 25 nmol/kg (corresponding to ~175-400 IU/kg FIX clotting activity) to FIX KO mice. Blood samples were collected starting at 5 min, and up to 336 h. FIX plasma levels were measured with an ELISA-based assay with anti-human FIX paired antibodies. PK was evaluated by non-compartmental analysis. Biodistribution: 3H-labeled recombinant FIXs were administered intravenously at doses of 200 IU/kg to FIX KO mice. Plasma levels and organ distribution were quantified starting at 15 min, and up to 240 h. Pharmacodynamics: FIX KO mice were treated intravenously with 50 IU/kg FIX clotting activity (nominal or labeled potency) of different rFIX products at 24, 72, 120 168 and 336 h prior to determination of bleeding time and total blood loss in a tail clip bleeding model. Immediately upon lesion, the tail tip was submerged in isotonic saline (0.9 %), kept at the mice physiological body temperature. Time to hemostasis is quantified as the time until bleeding stops for a minimum of 2 min. The volume of total blood loss was calculated by measuring the hemoglobin present in the isotonic saline solution at the end of the experiment. Results: Distinct PK profiles were observed for the three FIX molecules, where rFIX and rFIX-Fc exhibit an initial rapid distribution phase from plasma, while rFIX-FP showed a monophasic elimination profile up to 120 h post administration (p.a.). In the terminal phase, rFIX levels were quantifiable for up to 48 h p.a., while both; rFIX-FP and rFIX-Fc were measurable in plasma up to 240 h p.a. In line with this, the overall exposure AUC 0-inf is ranked in the following order: rFIX-FP > rFIX-Fc > rFIX. In the biodistribution study, a similar plasma PK profile was determined. Given the sensitivity of the radioactive method, an exposure plateau was observed for rFIX-Fc, and at lower levels for rFIX, whereas rFIX-FP continued to exhibit monophasic plasma clearance behavior. rFIX-FP exposure in the extravascular space (EVS) was lower than for the other FIX products. This is in line with volumes of distribution (Vss and Vz) which were highest for rFIX-Fc (AUC ranking rFIX-Fc > rFIX > rFIX-FP). FIX hemostatic efficacy in tail clip model was comparable for all 3 FIXs at the early time points but diverged at later time points post dosing. The blood loss and bleeding time measurements returned to baseline within 168 h for rFIX and rFIX-Fc, while the rFIX-FP group maintained robust hemostatic activity for up to 336 h. In contrast to lowest tissue exposure of rFIX-FP, the plasma AUC for rFIX-FP was highest, compared to FIX-Fc or FIX. In line, efficacy over time was also highest for rFIX-FP, suggesting that tissue exposure might not be the main driver for hemostatic activity. Conclusion: Different FIX products exhibit divergent PK and PD behaviors. rFIX-FP plasma PK profile suggests somewhat lower tissue distribution in comparison to rFIX-Fc and rFIX, which was confirmed in the tissue biodistribution study. Despite its limited extravasation into tissue, rFIX-FP exhibits superior and prolonged hemostatic activity in the FIX KO mouse tail clip model. rFIX and rFIX-Fc show comparable tissue biodistribution behavior, with robust extravasation into the EVS. Despite having the longest half-life and overall (plasma and tissue) exposure in the mouse, rFIX-Fc lost hemostatic activity in the tail clip model significantly faster than rFIX-FP. As a result, hemostatic efficacy was highest for the FIX-FP, the product with the lowest distribution volumes. The results therefore suggest that EVS is not the main determining factor for FIX efficacy in vivo. Disclosures Pestel: CSL Behring Innovation GmbH: Current Employment, Current equity holder in publicly-traded company. Peil: CSL Behring Innovation GmbH: Current Employment, Current equity holder in publicly-traded company. Knoll Machado: CSL Behring Innovation GmbH: Current Employment, Current equity holder in publicly-traded company. Claar: CSL Behring Innovation GmbH: Current Employment, Current equity holder in publicly-traded company. Raquet: CSL Behring Innovation GmbH: Current Employment, Current equity holder in publicly-traded company. Ponnuswamy: CSL Behring Innovation GmbH: Current Employment, Current equity holder in publicly-traded company. Mischnik: CSL Behring Innovation GmbH: Current Employment, Current equity holder in publicly-traded company. Herzog: CSL Behring GmbH: Current Employment, Current equity holder in publicly-traded company.

scholarly journals A Quantitative Systems Pharmacology (QSP) Model to Compare the Non-Clinical Biodistribution and Efficacy between Recombinant Factor IX (rIX) Therapies

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4240-4240
Author(s):  
Sabine Pestel ◽  
Douglas Chung ◽  
Alireza Rezvani-Sharif ◽  
Ineke Muir ◽  
Sivarmurthy Krupa ◽  
...  
Keyword(s):  
Blood Loss ◽  
Significant Role ◽  
Bleeding Time ◽  
Total Blood Loss ◽  
Publicly Traded ◽  
Total Blood ◽  
The Past ◽  
Binding Partners ◽  
Pharmacologically Active

Abstract Background and Aims: Replacement FIX therapy (rIX) is an effective treatment for hemophilia B even with undetectable levels in the blood 1. However, the mechanistic reason for hemostasis with low plasma levels is not well understood. There is growing evidence that FIX interactions with one or multiple binding partners (BP), may play a significant role in the exposure and hemostatic efficacy of rIX 2,3. The aim of this study is to explore this hypothesis by comparing the plasma PK, tissue biodistribution, and in vivo endpoints of different rIX variants using a mouse QSP model. Method:  in vitro and in vivo FIX-KO mice studies and mathematical models were used to build a QSP model consisting of 8 tissue compartments , with each tissue divided into vascular, endothelial and interstitial spaces 4,5. The model simulates endogenous mouse IgG (mIgG), mouse serum albumin (MSA), and rIX dynamics including key clearance and distribution mechanisms. Competition for the endothelial FcRn receptor between Fc, albumin, mIgG, and MSA is explicitly modeled 6,7,8. The model was calibrated using mouse studies of radiolabeled rIX-Fc (Alprolix®), rIX-WT (BeneFIX®), and rIX-FP (Idelvion®). Tail-clip experiments following administration of rIX-Fc, rIX-WT, and rIX-FP were used to correlate the predicted exposures with the observed effects on bleeding time and total blood loss. Results: Preliminary simulations proved that having at least one BP best explains the rapid distribution of rIX-Fc and rIX-WT into the tissues, and the long plasma T 1/2 of rIX-Fc and rIX-FP. Visual predictive checks of the full PBPK model showed good agreement with the PK in the tissues. The best fit was achieved using a specific arrangement of four distinct binding partners: Shared BP (SBP) between all compounds (e.g. N-terminal binder) located within the vasculature with estimated K D of 470/600/4100 nM, for rIX-WT/rIX-FP/rIX-Fc, respectively. BP binding specific to rIX-WT (e.g. C-terminal binder) located in the interstitium of the tissue (varying densities) with estimated K D of 23 nM BP binding only for rIX-FP (e.g. albumin binder) located in both; the vasculature and interstitium of the tissue with estimated K D 20/0.05 μM (vascular/interstitial) BP binding only for rIX-Fc (e.g. Fc binder) located in the interstitium of tissue (varying densities) with estimated K D 3 μM The high degree of extravasation of rIX-Fc (and rIX-WT to a lesser degree) results in rapid distribution and sequestration in the tissues. The limited extravasation of rIX-FP and its high affinity to the SBP, results in increased recovery and a greater pool of bound rIX available in the tissue vasculature. Additionally, strong inverse correlation between the bound rIX in the vasculature and bleeding time/total blood loss suggests that the vascular pool plays a more significant role in FIX pharmacology, as compared to the pool in the extravascular space. Conclusion: The mouse QSP model demonstrated that the plasma and tissue biodistribution of rIX-Fc, rIX-FP, and rIX-WT cannot be explained without a BP, and that it is plausible to assume that different binding partners, both intra- and extravascular, for different rFIX variants exist. The correlation between the levels of bound rIX and the coagulation endpoints suggests that the vascular bound rIX may be the pharmacologically active pool or reservoir for haemostasis. The extravasation and sequestration of rIX-WT and rIX-Fc into the tissues may explain the decreased vascular exposure, and hence, the reduced efficacy (increased bleeding time/total blood loss) at later time points. Although the exact identity of the BP's remains to be further elucidated, the model estimates of their affinity, density and location provide guidance for further experimental investigations. Expansion of the QSP model with additional data and coagulation kinetics will further our understanding of the role of BPs in rIX pharmacology. References 1Srivastava A et al (2013) Haemophilia 19(1), e1-47 2Feng D et al (2013) JTH, Vol. 11 (12), 2176-2178 3Cheung WF et al (1996) PNAS USA, 93(20), 11068-11073 4Li L et al (2014) AAPS Journal 16(5), 1097-1109 5Shah DK & Betts AM (2012) J Pharmacokinet Pharmacodyn 39(1), 67-86 6Chia J et al (2018) J Biol Chem 293(17), 6363-6373 7Andersen JT et al (2010) J Biol Chem 285(7), 4826-4836 8Andersen JT et al (2013) J Biol Chem 288(33), 24277-24285 Disclosures Pestel: CSL Behring Innovation GmbH: Current Employment, Current equity holder in publicly-traded company. Rezvani-Sharif: CSL Behring Ltd: Current Employment, Current equity holder in publicly-traded company. Muir: CSL Behring Ltd: Current Employment, Current holder of stock options in a privately-held company. Krupa: CSL Behring LLC: Current Employment, Current equity holder in publicly-traded company. Brechmann: CSL Behring Innovation GmbH, Ended employment in the past 24 months: Bayer Ag (Bayer Pharmaceuticals),: Current Employment, Ended employment in the past 24 months, Patents & Royalties: Bayer. Verhagen: CSL Behring Ltd: Current Employment, Current equity holder in publicly-traded company. Dower: CSL Behring Ltd: Current Employment, Current equity holder in publicly-traded company. Herzog: CSL Behring GmbH: Current Employment, Current equity holder in publicly-traded company.

Pre-Clinical Characteristics of a Recombinant Fusion Protein Linking Activated Coagulation Factor VII with Albumin (rVIIa-FP)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1115-1115
Author(s):  
Sabine Zollner ◽  
Daniel Schuermann ◽  
Elmar Raquet ◽  
Jochen Mueller-Cohrs ◽  
Thomas Weimer ◽  
...  
Keyword(s):  
Half Life ◽  
Animal Species ◽  
Hemophilia A ◽  
Procoagulant Activity ◽  
Factor Vii ◽  
Venous Stasis ◽  
Systemic Availability ◽  
Time Points ◽  
Activity Measurements ◽  
Hemostatic Activity

Abstract Abstract 1115 Recombinant factor VIIa (rFVIIa) is approved to control bleeding in hemophilia A and B patients who have developed inhibitory antibodies to replacement therapy and in acquired hemophilia. rFVIIa is rapidly eliminated with a terminal half-life of approximately 2.5 hours in humans. This short half life necessitates frequent injections and considerably limits prophylactic use. A recombinant fusion protein linking activated factor VII with albumin (rVIIa-FP) was engineered to extend the half life of rFVIIa. The present studies were conducted to gather knowledge about the pharmacokinetic and pharmacodynamic (PK/PD) properties of rVIIa-FP in preclinical animals models to enable a more precise estimate of the procoagulant activity of rVIIa-FP for the future clinical use in haemophilia patients with inhibitors to FVIII and FIX. Single intravenous doses of either rVIIa-FP or the licensed rFVIIa comparator, NovoSeven® were administered to mice, rabbits and monkeys. Subsequently, plasma samples were harvested at different time-points after study drug administration and systemic levels of rVIIa-FP or NovoSeven® were assessed either by FVIIa activity measurements using the Staclot® assay system or by FVII antigen measurements using an enzyme linked immunoabsorbance assay (ELISA). When investigating dose responses or duration of hemostatic activity, rVIIa-FP and NovoSeven® were administered to mice or rabbits before tail clip or induction of venous stasis, respectively. Following a single intravenous bolus application the procoagulant activity of both study drugs was assessed in both models at different time points after administration up to 24 hours to proof prolonged procoagulant effects of rVIIa-FP compared to NovoSeven®. Intriguingly, in all animal species tested the systemic bioavailability, clearance and in addition the terminal half-life (t1/2β), of rVIIa-FP were significantly better in comparison to NovoSeven®. Based on FVIIa activity measurements in plasma samples derived from monkeys the 11 fold higher area under the curve (AUC) was accompanied with a 4-fold longer t1/2β, while in vivo recovery (IVR) of rVIIa-FP exceeded that of NovoSeven® by a 70 %. In rabbits the 20 fold higher AUC was associated with a 5-fold longer t1/2β and a 4-fold higher IVR when measuring specific FVIIa activity. In mice measurements of FVII antigen revealed a 11 fold higher AUC, while t1/2β was 4 times longer and IVR was found to be doubled in comparison to NovoSeven®. The assessment of pharmacodynamic activity after dosing based on specific FVIIa activity adjusted by the Staclot® assay revealed, that the procoagulant effect of rVIIa-FP was not different from NovoSeven®, when measuring total blood loss under acute bleeding conditions after tail clip. However, when assessing late time-points after treatment the prolonged systemic availability of rVIIa-FP translated into a longer and sustained hemostatic activity of rVIIa-FP compared to NovoSeven® in both animal models. Intriguingly, ex vivo studies in hemophilia A mice showed that the 4 fold prolonged systemic availability of rVIIa-FP in plasma translates into sustained FVIIa activity when recorded as thrombin generation activity compared to NovoSeven® at 16 hours after treatment. Furthermore in rabbits, the 5 fold longer half-life translated into a significantly greater procoagulant activity as measured by thrombus formation in both jugular veins after venous stasis. In conclusion, the recombinant albumin fusion technology was successfully applied to human recombinant FVIIa for a significantly improved of PK/PD profile as observed in different pre-clinical animal species. Future clinical studies can proof whether the observed improved PK/PD characteristics will also translate into a half-life extension and a longer hemostatic effect in hemophilia patients with inhibitors to FVIII and FIX. Allowed: The entire body of the abstract, including text and tables, must not exceed 3,800 characters. Spaces are not included in this number; title, authors' names, and affiliations are counted separately. Figures are not included in the character count. Disclosures: Zollner: CSL Behring GmbH: Employment. Schuermann:CSL Behring GmbH: Employment. Raquet:CSL Behring GmbH: Employment. Mueller-Cohrs:CSL Behring GmbH: Employment. Weimer:CSL Behring GmbH: Employment. Pragst:CSL Behring GmbH: Employment. Dickneite:CSL Behring GmbH: Employment. Schulte:CSL Behring GmbH: Employment.

Studies on the Action of Ethamsylate (Dicynene) on Haemostasis

1986 ◽  
Vol 56 (01) ◽  
pp. 006-008 ◽  
Author(s):  
R A Hutton ◽  
E A Wickham ◽  
J V Reed ◽  
E G D Tuddenham
Keyword(s):  
Single Dose ◽  
Platelet Aggregation ◽  
Blood Loss ◽  
Plasma Levels ◽  
Healthy Subjects ◽  
Bleeding Time ◽  
Secondary Wave ◽  
Double Blind Trial ◽  
Double Blind ◽  
Platelet Defects

SummaryTwelve healthy subjects received ethamsylate or a placebo by mouth over a 48 h period in a randomized double-blind trial. The template bleeding time (including estimation of amount of blood loss), platelet aggregation studies, and plasma levels of plasminogen, alpha2-antiplasmin and fibronectin were carried out before and during treatment. The effect of a single dose (600 mg) of aspirin, given 24 h after commencement of treatment, was also determined.Neither ethamsylate nor placebo caused a significant change in the basal values of any of the variables monitored but both the prolongation of the bleeding time and the amount of blood loss induced by aspirin were significantly less during ethamsylate treatment than with placebo. Ethamsylate failed to prevent the aspirin-induced elimination of the secondary wave of platelet aggregation.We conclude that ethamsylate may reduce the haemorrhagic symptoms associated with mild functional platelet defects.

scholarly journals Cholinergic Stimulation Improves Hemostasis in a Hemophilia Mouse Model

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3528-3528 ◽  
Author(s):  
Shilpa Shukla ◽  
Jason R. Fritz ◽  
Crystal A. Kyaw ◽  
Sara P. Valentino ◽  
Catherine W. Imossi ◽  
...  
Keyword(s):  
Blood Loss ◽  
Vagus Nerve ◽  
Nerve Stimulation ◽  
Vagus Nerve Stimulation ◽  
Thrombin Generation ◽  
Bleeding Time ◽  
Hemophilia A ◽  
Cholinergic Stimulation ◽  
Total Blood ◽  
Traumatic Hemorrhage

Abstract Background: Management of bleeding in hemophilia patients that develop inhibitors remains a challenging clinical problem. Current bypassing agents are expensive, not universally effective and have pro-thrombotic potential. New approaches, including bispecific antibodies and others have shown potential but still require additional testing in clinical trials. As such, there is a clear need for alternative therapeutic options. The inflammatory reflex is an endogenous neuro-immune pathway that monitors and regulates systemic inflammation via the vagus nerve. The afferent vagus nerve can inform the brain of increased peripheral inflammation. The brain then down-regulates systemic inflammation via increased efferent vagus nerve signaling to the spleen, a mechanism referred to as the cholinergic anti-inflammatory pathway (KJ Tracey, Nat Rev Imm 9(6), 2009). Our previous work has demonstrated that activation of the cholinergic anti-inflammatory pathway via direct electrical vagus nerve stimulation (VNS) or by systemic administration of a pharmacological cholinergic agonist inhibits proinflammatory cytokine production and protects against lethal systemic inflammation (JR Fritz, Bioelectron Med 1(1), 2014). While studying the cholinergic anti-inflammatory pathway in mice, we observed that cholinergic stimulation reduces traumatic hemorrhage. To explore this observation further, we developed a porcine model of soft tissue trauma and hemorrhage. Electrical stimulation of the cervical vagus nerve significantly reduces total blood loss and bleeding time in pigs. Electrical vagus nerve stimulation also significantly increases local thrombin generation at the site of injury, whereas systemic thrombin concentrations remain unchanged (CJ Czura, Shock 33(6), 2010). Aim: To evaluate the beneficial hemostatic effects of cholinergic stimulation on tail hemorrhage in a mouse model of hemophilia A. Methods: Male 8-12-week-old, factor VIII knockout mice (B6;129S-F8<tm1Kaz>/J, Jackson Labs) receive either electrical cervical vagus nerve stimulation (1 V, 30 Hz, 2 ms pulse for 5 minutes) or sham stimulation under anesthesia (ketamine/xylazine 140/16 mg/kg, ip) five minutes before 2 mm distal tail transection. In separate experiments, nicotine (2 mg/kg, ip) or saline is administered 17 minutes before injury. Mouse tails are placed into a 37¡ÆC normal saline bath for five minutes prior to injury. After injury, tails are placed into 50 mL conical tubes filled with 37¡ÆC normal saline and allowed to bleed freely for a total of ten minutes. Total shed blood volume and active bleeding time are recorded, and systemic and local thrombin generation (thrombin-antithrombin complex) is measured via ELISA. Complete blood counts are measured via standard clinical assay. Results: Vagus nerve stimulation significantly reduces total blood loss and bleeding time by 75% and 18%, respectively. Administration of nicotine significantly reduces total blood loss and bleeding time by 79% and 41%, respectively (Fig. 1). Nicotine significantly increases local thrombin generation compared with vehicle controls, whereas systemic thrombin generation is unchanged. There are no differences in circulating platelet counts or hematocrit levels. Conclusions: Cholinergic stimulation results in rapid and specific improvements in hemostasis during traumatic hemorrhage in a mouse model of hemophilia A. The molecular mechanisms of localized thrombin generation remain an active area of research. Future clinical trials are necessary to determine if cholinergic stimulation is an efficacious, safe and cost-effective therapy for hemophilia A patients. Figure 1. Vagus nerve stimulation (A) and nicotine administration (B) significantly reduce blood loss following traumatic hemorrhage in hemophilia A mice. Figure 1. Vagus nerve stimulation (A) and nicotine administration (B) significantly reduce blood loss following traumatic hemorrhage in hemophilia A mice. Figure 2. Nicotine administation significantly increases thrombin generation specifically at the site of injury and not systemically. Figure 2. Nicotine administation significantly increases thrombin generation specifically at the site of injury and not systemically. Disclosures No relevant conflicts of interest to declare.

Selective Alteration of FXa Zymogenicity Provides a Dynamic Range of Variants that Improve Hemostasis in Hemophilia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 201-201
Author(s):  
Lacramioara Ivanciu ◽  
Rodney M. Camire
Keyword(s):  
Biological Activity ◽  
Blood Loss ◽  
Active Site ◽  
Half Life ◽  
Thrombin Generation ◽  
General Mechanism ◽  
New Class ◽  
Tail Clip

Abstract Abstract 201 There is significant interest in developing safe and effective procoagulant hemostatic molecules to treat bleeding disorders. Recently, our laboratory has generated a novel FXa variant (e.g. FXaI16L) with zymogen-like properties that shows promise in bypassing deficiencies upstream of the common pathway. FXaI16L is rendered partially inactive due to a defect in transitioning from zymogen to protease where residue 16 is critical to this process. However, the biological activity of FXaI16L is rescued once its cofactor FVa is made available following coagulation activation. While FXaI16L is effective in correcting the hemophilia phenotype both in vitro and in vivo, the mutation site and general mechanism of action indicates that there is inherent flexibility in this new class of bypassing agent. To this end, we extended the mutational framework at positions 16 and 17 and created a series of zymogenic mutants exhibiting a broad range of activities. We surmised that depending on the amino acid at these positions, the transition from zymogen to protease will be altered to varying degrees. Following an initial screen of ten variants, five were pursued which fell into three categories relative to wild-type (wt)-FXa: “zymogen-like 1” (<1% activity), “zymogen-like 2” (∼3% activity; similar to FXaI16L with ∼5% activity) and “zymogen-like 3” (∼20% activity). Kinetic studies revealed that all mutants have an impaired ability to bind a small chromogenic substrate at the active site with zymogen-like 1 variants having the weakest affinity. Since physiological inhibitors of FXa predominantly target the active site, half-lives of the variants should correlate with zymogenicity. In line with this, zymogen-like 1 variants exhibited the longest half-life (3–4 hr) in hemophilia B (HB) plasma, followed by zymogen-like 2 (1–2 hr) and zymogen-like 3 (0.25 hr); these are all much greater than wt-FXa (<1–2 min). Assessment of biological activity in HB plasma by aPTT revealed that while wt-FXa (0.1 nM) normalized the prolonged clotting time, the zymogen-like variant 3 was ∼75% effective and zymogen-like 2 derivatives were ∼50% effective. Surprisingly, even the zymogen-like 1 variants with almost undetectable chromogenic activity, shortened the prolonged aPTT (<20% effective). However, the apparent activities of each zymogenic mutant were greatly improved when saturating amounts of FVa were added, indicating that suboptimal amounts of the cofactor are generated during the clotting assay. Furthermore, in a thrombin generation assay using HB plasma each zymogenic variant (0.1 nM) yielded a robust IIa signal with endogenous IIa potential (ETP) and peak heights comparable to wt-FXa. Only the zymogen-like 1 variant had a prolonged time to peak thrombin (∼25 min vs. ∼15 for all other variants/wt-FXa) indicating a delay in thrombin generation. We speculate this reflects the time needed to increase the FVa concentration during the assay, as variants with enhanced zymogen-like character require higher concentrations of cofactor to rescue catalytic function. Next, we evaluated whether these in vitro parameters had a correspondingly relevant impact on the hemophilic phenotype in vivo. In a tail clip assay two different experiments were conducted in which proteins were infused into HB mice (n = 3–6; 450 μg/kg) either 2 min after or 5 min prior to injury. In either experiment, total blood loss in HB mice treated with zymogen-like 1 or zymogen-like 2 variants was significantly reduced compared to PBS treated (n = 5) animals. Furthermore, in a third experiment (n = 3–6; 450 μg/kg) in which proteins were infused 30 min prior to injury only zymogen-like 1 variants were effective in reducing blood loss. Counterintuitively, the ‘higher' activity zymogen-like 3 variant was not particularly effective in the tail clip model. Collectively these in vivo data show that the more active the protease the more difficult it is to overcome the protective mechanism of circulating inhibitors (e.g. antithrombin III) to achieve a therapeutic benefit. By evading interactions with inhibitors and imparting a prolonged half-life, the new zymogenic variants may have a sustained impact on improving hemostasis in hemophilia. We conclude that the plasticity of this new class of FXa variants provides a useful and flexible platform to selectively bioengineer activity and half-life. Disclosures: Camire: Pfizer: Patents & Royalties, Research Funding.

Detection of Carriers of Hemophilia by Hemorrhagometry

1975 ◽  
Author(s):  
J. Jesdinsky-Buscher ◽  
A. H. Sutor ◽  
H. Niederhoff
Keyword(s):  
Blood Loss ◽  
Cold Tolerance ◽  
Room Temperature ◽  
Bleeding Time ◽  
Hemophilia A ◽  
Tolerance Test ◽  
Normal Range ◽  
Total Blood ◽  
Abnormal Bleeding

Hemorrhagometry, a new in-vivo method, measures bleeding time, intensity and total blood loss from a small standardized wound (Sutor et al.: Blut 22, 27, 1971). In patients with hemophilia these values are within the normal range when the test is performed at room temperature (24° C). However, when the wound is cooled to 17° C (cold tolerance test), bleeding time is abnormally long in hemophiliacs. Therefore, we investigated this test in carriers of hemophilia. The cold tolerance test was performed in 16 ‘proven’ and 6 ‘probable’ carriers (criteria according to Nilsson). 14 proven and 4 probable carriers showed abnormal bleeding times of 15 min and more (2 proven carriers of hemophilia B did not differ markedly from 20 carriers of hemophilia A). When hemorrhagometry was performed at room temperature, the carriers could not be distinguished from normal control persons.Of 15 sisters and aunts of hemophiliacs 9 had abnormal cold tolerance test findings, in fair agreement with the probability of .5 to be expected theoretically. Thus the hemorrhagometry cold tolerance test is helpful in detecting carriers of hemophilia.

scholarly journals VWF-FVIII Interactions Influence Hemostatic Thrombus Stability in Murine Models of Hemophilia Α and Type 2N VWD

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1403-1403
Author(s):  
Laura L Swystun ◽  
Courtney Dwyer ◽  
Kate Nesbitt ◽  
Kassandra Hebert ◽  
David Lillicrap
Keyword(s):  
Blood Loss ◽  
Research Funding ◽  
Bleeding Time ◽  
Thrombus Formation ◽  
Wild Type ◽  
Bleeding Events ◽  
Experimental Conditions ◽  
Total Blood ◽  
Tail Vein ◽  
Deficient Mice

Abstract von Willebrand factor (VWF) and factor VIII (FVIII) circulate in the plasma as a non-covalent complex. VWF can influence FVIII activity by stabilizing FVIII plasma levels and transporting FVIII to the site of thrombus formation. We have previously generated a murine model of type 2N VWD through hydrodynamic expression of the severe R816W VWF variant that exhibits a 90% decrease in FVIII-binding ability and a failure to stabilize endogenous FVIII when expressed in a VWF-deficient mouse. Intravital studies of arteriole platelet thrombus formation demonstrated that FVIII knockout and type 2N VWD mice produced smaller, less stable thrombi. Aim: In this study, we assess the contribution of VWF and FVIII interactions to the formation and stability of hemostatic thrombi in a murine tail vein transection (TVT) bleeding model. Method: Under isoflurane anaesthesia the left lateral tail vein was transected using standardized measuring and transection templates (provided by Novo Nordisk, described in detail in Johansen et al. Hemophilia. 2016). Bleeding time was observed over the course of 60 minutes. Blood was collected into saline for hemoglobin quantification. For experimental conditions, 300 U/Kg wild type or type 2N (R816W) VWF was administered IV to VWF KO mice 2 hours prior to TVT in order to allow for endogenous FVIII stabilization. Results: Consistent with previous reports, VWF (44.15 min; p<0.0001) and FVIII-deficient mice (45 min; p<0.0001) exhibited a significantly prolonged total bleeding time relative to normal mice (4.6 min). Infusions of plasma-derived murine VWF (FVIII-free) into VWF deficient mice have demonstrated that wild type (WT) VWF is capable of restoring FVIII:C levels to ~75% 2 hours post-infusion, while the R816W variant does not influence FVIII stabilization and levels remain at ~15%. Mice infused with WT VWF had a shorter bleeding time (16.2 min) relative to mice infused with the severe type 2N VWD variant (44.67 minutes, p<0.0001). For FVIII KO mice, the primary bleeding time (1.53 min), defined as the period of time until first bleeding cessation, was not different from normal mice (1.2 min) but significantly extended for VWF KO mice (35.97 min, p<0.0001). Primary bleeding time was markedly reduced for VWF KO mice infused with WT VWF (4.97 min, p=0.07) confirming the restoration of primary hemostasis by infusion of murine plasma-derived VWF. Hemoglobin quantification confirmed that FVIII (243.8 mg, p<0.0001) and VWF KO mice (198.8 mg, p<0.0001) had increased blood loss compared to normal mice (9.91 mg). Similarly, VWF KO mice infused with type 2N VWF (308.04 mg) had increased blood loss compared to mice infused with WT VWF (73.78 mg, p=0.0008). Total blood loss and bleeding times were positively correlated across all groups (r2=0.625, p<0.0001). Thrombus stability was characterized by the frequency of spontaneous re-bleeding events. 11% of normal mice experienced one or more spontaneous re-bleeding events during the course of injury, while 100% of FVIII deficient mice experienced spontaneous re-bleeding (p=0.0004). A similar trend was observed for VWF KO mice infused with WT VWF (0%) and type 2N VWF (66.7%, p=0.021). Conclusions: These studies suggest that in the murine TVT model of physiological hemostasis, the initial hemostatic thrombus formation is predominantly driven by platelet plug formation and is VWF-dependent. In contrast, thrombus stability over the course of the model is reliant on the processes responsible for fibrin formation, and is FVIII-dependent. This suggests that patients with type 2N VWD and hemophilia A may have bleeding that is the result of impaired stabilization of the primary platelet plug. Disclosures Lillicrap: Baxalta: Research Funding; Biogen-Idec: Research Funding; Bayer: Research Funding; Octapharma: Research Funding.

DOES DESMOPRESSIN ACETATE REDUCE BLOOD LOSS AFTER CARDIOPULMONARY BYPASS SURGERY?

1987 ◽  
Author(s):  
E Rocha ◽  
R Llorens ◽  
J A Paramo ◽  
R Arcas ◽  
B Cuesta ◽  
...  
Keyword(s):  
Placebo Group ◽  
Cardiopulmonary Bypass ◽  
Blood Loss ◽  
Bypass Surgery ◽  
Bleeding Time ◽  
Prospective Trial ◽  
Reduce Blood Loss ◽  
Total Blood ◽  
Cardiopulmonary Bypass Surgery ◽  
Transfusion Requirements

It has been suggested that desmopressin acetate (DDAVP) administration reduces blood loss after cardiac surgery. We have investigated the effect of DDAVP administration in a doubleblind, randomized, prospective trial including 60 patients undergoing cardiopulmonary bypass surgery. Thirty patients received 0.3 ug/kg DDAVP and 30 patients a placebo. The infusion was administered in a 50 ml saline solution over 15 min when cardiopulmonary bypass had been concluded. Blood samples were taken before surgery, immediately before and 90 min after DDAVP or placebo administration, and 24 hours postoperatively. The following parameters were measured in each sample: hematocrit, hemoglobin, platelet count, VIII:C and factor VIII:vWF. Bleeding time was also measured before operation and 90 min after treatment administration. Blood loss and transfusion requirements were evaluated from the beginning of treatment until 72 hours after surgery. Results showed no significant differences neither in total blood loss (833 ± 363 ml in the DDAVP group vs. 907 ± 646 in the placebo group) nor in blood transfusion (1633 ± 676 ml in the DDAVP group vs. 1643 ± 720 in the placebo group). The prolongation of bleeding time and the decrease of factor VIII:vWF, 90 min after treatment, were significantly lower (p < 0.05) in the DDAVP group as compared with the placebo group. We conclude that DDAVP administration does not reduce blood loss in patients undergoing cardiopulmonary bypass surgery, which would suggest a more complex mechanism to explain the increased bleeding in these patients.

Plasma Thrombospondin as an Indicator of Intravascular Platelet Activation in Patients with Vasculitis

1987 ◽  
Vol 58 (03) ◽  
pp. 850-852 ◽  
Author(s):  
M B McCrohan ◽  
S W Huang ◽  
J W Sleasman ◽  
P A Klein ◽  
K J Kao
Keyword(s):  
Platelet Activation ◽  
Plasma Levels ◽  
Half Life ◽  
Degradation Products ◽  
Plasma Concentrations ◽  
Primary Source ◽  
Positive Correlation ◽  
Activation Marker ◽  
Fibrin Degradation Products ◽  
The Mean

SummaryThe use of plasma thrombospondin (TSP) concentration was investigated as an indicator of intravascular platelet activation. Patients (n = 20) with diseases that have known vasculitis were included in the study. The range and the mean of plasma TSP concentrations of patients with vasculitis were 117 ng/ml to 6500 ng/ml and 791±1412 ng/ml (mean ± SD); the range and the mean of plasma TSP concentrations of control individuals (n = 33) were 13 ng/ml to 137 ng/ml and 59±29 ng/ml. When plasma TSP concentrations were correlated with plasma concentrations of another platelet activation marker, β-thromboglobulin (P-TG), it was found that the TSP concentration inei eased exponentially as the plasma β-TG level rose. A positive correlation between plasma levels of plasma TSP and serum fibrin degradation products was also observed. The results suggest that platelets are the primary source of plasma TSP in patients with various vasculitis and that plasma TSP can be a better indicator than β-TG to assess intravascular platelet activation due to its longer circulation half life.

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