Hemostatic Potential Research Articles

Difference in Fibrin Clot Structure in Haemophilia A and Haemophilia B

Introduction: Hemophilia A and Hemophilia B are X-linked bleeding disorders characterized by deficiency of coagulation factor VIII and IX respectively. The disorders have traditionally been considered to display identical symptoms. However, recent clinical data suggest that hemophilia B has a less severe phenotype than hemophilia A in patients with similar levels of their deficient factor. There is a distinct lack of laboratory studies comparing the hemostatic potential in patients with the same severity grade of Hemophilia A and Hemophilia B. We assessed fibrin clot quality in Hemophilia A and Hemophilia B patient plasma as a possible explanation to the milder bleeding phenotype in patients with Hemophilia B. To achieve this a liquid permeation technique was used, which has been used to investigate fibrin networks in previous in vitro studies (He et. al. Blood Coagul Fibrinolysis 2005).Methods: 20 patients with hemophilia A and 28 with hemophilia B were considered for inclusion in the study. Patients were recruited from centers in Stockholm and Belgrade and were thereafter matched according to their deficient factor level. Hemophilia diagnosis and informed consent formed the basis for study inclusion and exclusion criteria were lack of consent and inability to provide a matched patient from the other group. 17 hemophilia A and 17 hemophilia B patients were included in the study. The patient matching according to factor level was tested with Spearman’s rank-order correlation, yielding a correlation coefficient of 0.999 (p<0.001).In vitro fibrin clots were created from centrifugated patient plasma, CaCl2 and bovine thrombin. They were then permeated with a percolating buffer with a pre-determined viscosity (Tris-hydroxymethyl aminomethane, imidazole, NaCl and trasylol) under three different pre-determined hydrostatic pressures. Fibrin clot permeability was calculated in Darcy constants (Ks) using the equation: Ks = (Q x L x h) / (t x A x ΔP). where Q is the elution volume (cm3), t is the buffer percolation time (seconds), h is the viscosity (poise, dyne x s cm-2), L is the fibrin clot length (cm), A is the area of the fibrin clot (cm2) and ΔP is the difference of pressure (dyne cm-2). A high Ks represents high permeability, which is interpreted as porous fibrin clots. In contrast low Ks values represent low permeability, which is interpreted as low porosity.Results: Ks was higher in hemophilia A than in hemophilia B. Mean Ks for hemophilia A was 12.28 ± 5.92 and for hemophilia B 6.16 ± 3.63 (p<0.0005). Subanalysis of moderate and severe hemophilia patients (8 matched pairs, 16 patients) showed higher mean Ks in hemophilia A. Mean Ks of the hemophilia A group was 14.90 ± 6.88 and for the hemophilia B group 5.78 ± 3.86, p=0.039.Conclusion: Fibrin clot structure exhibits less permeability in hemophilia B than hemophilia A, which may be one of the explanations to the milder bleeding symptoms observed in hemophilia B at the same level of deficient factor. This finding is in accordance with clinical studies showing that HB has a less severe bleeding phenotype than HA. DisclosuresAntovic:Baxter Healthcare Corporation: Honoraria, Research Funding; Siemens: Honoraria; Stago: Honoraria; Sysmex: Honoraria; Novo Nordisk: Honoraria; Roche: Honoraria. Chaireti:Baxalta: Research Funding.

Silk fibroin based carrier system for delivery of fibrinogen and thrombin as coagulant supplements.

The control of bleeding is one of the most important interventions after a traumatic injury. Hemostatic devices delivering blood clotting accelerating agents such as fibrinogen are increasingly used due to their efficacy and their ease of application. In the present study, we describe a method to incorporate the coagulant supplements fibrinogen and thrombin in silk protein sponges by mixing the coagulants with an aqueous silk solution, followed by molding, freeze-drying, and water annealing. In this combination system, we demonstrate the delivery of fibrinogen while maintaining its hemostatic potential. Concentration ratios of silk to fibrinogen of 1.0%/2.8%, 2.3%/1.5%, and 3.0%/0.8% were used. The thrombin-induced fibrin polymeric network filled the space in and next to the silk spongy structure but also remained interconnected to the silk, providing an intact network. The mechanical characterization of the fibrinogen-releasing silk sponges before and after the induction of the fibrinogen polymerization demonstrated that the fibrin network resulted in reduced permanent deformation from 21.1% to 6.5%, 19.6% to 5.7%, and 12.7% to 9.4% for the 2.8%, 1.5%, and 0.8% fibrinogen-containing silk sponges, respectively. Moreover, the fibrin formation lead to a more linear elastic behavior over longer strain ranges. In combination, the Calcein-AM/PI staining and MTT assay results indicate uniform cell adhesion on the surface and cytocompatibility of the silk/fibrin sponges, respectively. Moreover, the co-delivery of thrombin with fibrinogen via silk as carrier material is described, offering a more mechanically robust and durable system while preserving hemostatic features of the coagulant substances for the generation of hemostatic devices. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 687-696, 2017.

Enhanced pro-coagulant hemostatic agents based on nanometric zeolites

Micro-and nanometric faujasite zeolites were tested as hemostatic coagulant agents. The as-made zeolites and their calcium ion exchanged derivatives were characterized by XRD, SEM, AFM, TGA, and DSC. Zeta potential measurements of the micro and nanometric zeolites, as a function of pH (1–12.5), revealed the hemostatic potential of the materials. The isoelectric point for FAU, FAU/Ca, Nano-FAU, and Nano-FAU/Ca were measured at pH 2.0, 1.9, 3.2, and 2.5, respectively. The hemostatic activity was confirmed by the TEG technique for both micro and nanometric zeolites, however a superior activity were observed for the nanometric materials. FAU and FAU/Ca exhibited a reduction of the R parameter (defined as period of time of latency from the start of test to initial fibrin formation for the formation of a clot of an amplitude of 2 mm) from 8.6 ± 0.7 min (control) to 3.2 ± 0.7 min and 2.3 ± 0.1 min, respectively. On the other hand, for the Nano-FAU, and Nano-FAU/Ca the observed decrease were to 2.4 ± 0.6 min, and 1.1 ± 0.2 min, respectively. The amounts of heat released were 105.60 J/g (FAU), 65.8 J/g (FAU/Ca), 85.48 J/g (Nano-FAU), and 78.21 J/g (Nano-FAU/Ca). Statistical analysis using one-way analysis of variance showed a global value of p < 0.0001. Tukey-Kramer multiple comparisons revealed significant (p < 0.0001) differences for FAU, FAU/Ca, Nano-FAU, and Nano-FAU/Ca, relative to the control. Nano-FAU/Ca showed the most important reductions of the R with significant differences compared to the control (p < 0.0001 and p < 0.0001, respectively) and FAU (p = 0.0071 for the R parameter).

Upon admission coagulation and platelet function in patients with thermal and electrical injuries

RationalThere has been increased focus on hemostatic potential and function in the initial assessment of the patient with traumatic injuries, that not been extensively studied in patients with burns. We proposed to determine the hemostatic potential of patients with burns upon admission to the emergency department and contrasted their condition with that of healthy controls and patients with other traumatic injuries. In addition we assessed differences due to thermal versus electrical injury and evaluated the effect of burn size. MethodsThis is a patient based prospective observational study conducted with delayed consented. Subjects at the highest level of trauma activation upon admission to the ED had a blood sample collected for research purposes and were subsequently consented. Hemostatic potential was measured by rapid thromelastography (r-TEG®), thrombin generation by calibrated automated thrombogram (CAT) and platelet function by Multiplate® using five activators. Burn subjects were compared to subjects with other traumatic injuries and controls. Within the burn subjects additional analysis compared mechanism (thermal vs. electrical) and burn size. Values are medians (IQR). ResultsTwo hundred and eighty two trauma patients (with burns n=40, 14%) and 27 controls were enrolled. Upon admission, compared to controls, subjects with burns or trauma were hyper-coagulable based on r-TEG and CAT, with increased rates of clot formation and thrombin generation. There were no differences in burns compared to other traumatic injuries. The presence of hyper-coagulation did not appear to be related to the type of burn or the percentage of total body surface area involved. Employing previous defined cut points for R-TEG driven therapeutic interventions burn patients had similar rates of hyper- and hypo-coagulation noted in patients with traumatic injuries. ConclusionUpon admission patients with burns are in a hyper-coagulable state similar to that of other trauma patients. Employing demonstrated cut points of hemostatic potential in trauma patients associated with increased risk of poor outcomes demonstrated the incidence in burn patients to be similar, suggesting that these values could be used in the early assessment of the patient with burns to guide treatment interventions.

Design of Acacia Gum–Carbopol–Cross-Linked-Polyvinylimidazole Hydrogel Wound Dressings for Antibiotic/Anesthetic Drug Delivery

Keeping in view the antimicrobial nature of polyvinylimidazole, the antioxidant property of gum acacia, the mucoadhesive and gel-forming nature of Carbopol, the anesthetic action of lidocaine, and the antimicrobial action of gentamicin, in the present work, an attempt was made to prepare lidocaine and gentamicin loaded gum acacia–Carbopol–polyvinylimidazole based hydrogel wound dressings for better wound healing. These hydrogel dressings were characterized by 13C NMR, FTIR, SEM, cryo-SEM, AFM, XRD, DSC, swelling, and network parameter studies. Some properties of wound dressings were also determined which include the following: hemolysis, hemostatic potential, oxygen permeability, water vapor transmission rate, antioxidant activity, mucoadhesion, protein adsorption, degradation studies, antimicrobial properties (against Pseudomonas aeruginosa bacteria), microbial penetration, mechanical properties (tensile strength, burst strength, resilience, relaxation, folding endurance and gel strength) drug release, a...

Degradation and hemostatic properties of polyphosphate coacervates

Sodium polyphosphate is a linear polymer formed from phosphate units linked together by sharing oxygen atoms. Addition of calcium to a solution of sodium polyphosphate results in phase separation and formation of a polyphosphate coacervate best described as a polymeric rich viscoelastic material. Polyphosphate coacervate is an interesting candidate as a biomaterial based on its ability to bind with different cations and to be loaded with drugs. Here, in vitro degradation and hemostatic properties of polyphosphate coacervates are comprehensively evaluated. We show that polyphosphate coacervates degrade and dissolve at a fast rate, losing half of their original mass in a week and transforming to mainly pyrophosphate after 4weeks. This burst dissolution phase happens earlier for the coacervate prepared from very short chain polyphosphate but overall using longer polyphosphate chains does not increase the coacervate longevity significantly. Substitution of Ca with Sr or Ba does not affect the hydrolysis of coacervates but slows down their dissolution into the media. In a whole blood clotting assay, coacervates profoundly decrease the clotting time especially when very long chain polyphosphates are used. While coacervate chain length and divalent cation type were found to significantly affect prothrombin time and thromboplastin time compared to the control, no discernible trends were observed. Platelets adhere in large numbers to coacervates, especially those containing long chain polyphosphate, but the cell morphology observed suggests that they might not to be fully activated. Overall, the long chain polyphosphate coacervate holds a great potential as a resorbable hemostatic agent. Statement of SignificanceDivalent cation additions to a sodium polyphosphate solution result in polyphosphate coacervates, or highly viscous gel-like materials, having great potential in bio-applications such as drug delivery and hemostasis. As these coacervates degrade in aqueous environments, we undertook a comprehensive evaluation to better understand the impact of polyphosphate chain length and divalent cation substitution on this hydrolytic response in order to better predict degradation behavior in the body. Furthermore, there is great interest in the role of polyphosphates in hemostasis following recent publications showing that platelets secrete polyphosphates upon thrombin stimulation. In this paper, we evaluate the hemostatic potential of polyphosphate coacervates as bulk constructs, demonstrating that indeed these materials hold great potential as a degradable hemostatic agent.

Platelets regulate vascular endothelial stability: assessing the storage lesion and donor variability of apheresis platelets.

In current blood banking practices, platelets (PLTs) are stored in plasma at 22°C, with gentle agitation for up to 5 days. To date, the effects of storage and donor variability on PLT regulation of vascular integrity are not known. In this study, we examined the donor variability of leukoreduced fresh (Day 1) or stored (Day 5) PLTs on vascular endothelial barrier function in vitro and in vivo. In vitro, PLT effects on endothelial cell (EC) monolayer permeability were assessed by analyzing transendothelial electrical resistances (TEER). PLT aggregation, a measure of hemostatic potential, was analyzed by impedance aggregometry. In vivo, PLTs were investigated in a vascular endothelial growth factor A (VEGF-A)-induced vascular permeability model in NSG mice, and PLT circulation was measured by flow cytometry. Treatment of endothelial monolayers with fresh Day 1 PLTs resulted in an increase in EC barrier resistance and decreased permeability in a dose-dependent manner. Subsequent treatment of EC monolayers with Day 5 PLTs demonstrated diminished vasculoprotective effects. Donor variability was noted in all measures of PLT function. Day 1 PLT donors were more variable in their effects on TEER than Day 5 PLTs. In mice, while all PLTs regardless of storage time demonstrated significant protection against VEGF-A-induced vascular leakage, Day 5 PLTs exhibited reduced protection when compared to Day 1 PLTs. Day 1 PLTs demonstrated significant donor variability against VEGF-A-challenged vascular leakage in vivo. Systemic circulating levels of Day 1 PLTs were higher than those of Day 5 PLTs In vitro and in vivo, Day 1 PLTs are protective in measures of vascular endothelial permeability. Donor variability is most prominent in Day 1 PLTs. A decrease in the protective effects is found with storage of the PLT units between Day 1 and Day 5 at 22°C, thereby suggesting that Day 5 PLTs are diminished in their ability to attenuate vascular endothelial permeability.

Microparticles variability in fresh frozen plasma: preparation protocol and storage time effects.

Extracellular vesicles or microparticles exhibiting procoagulant and thrombogenic activity may contribute to the haemostatic potential of fresh frozen plasma. Fresh frozen plasma was prepared from platelet-rich plasma at 20 °C (Group-1 donors) or directly from whole blood at 4 °C (Group-2 donors). Each unit was aseptically divided into three parts, stored frozen for specific periods of time, and analysed by flow cytometry for procoagulant activity immediately after thaw or following post-thaw storage for 24 h at 4 °C. Donors' haematologic, biochemical and life-style profiles as well as circulating microparticles were analysed in parallel. Circulating microparticles exhibited a considerable interdonor but not intergroup variation. Fresh frozen plasma units were enriched in microparticles compared to plasma in vivo. Duration of storage significantly affected platelet- and red cell-derived microparticles. Fresh frozen plasma prepared directly from whole blood contained more residual platelets and more platelet-derived microparticles compared to fresh frozen plasma prepared from platelet-rich plasma. Consequently, there was a statistically significant difference in total, platelet- and red cell-derived microparticles between the two preparation protocols over storage time in the freezer. Preservation of the thawed units for 24 h at 4 °C did not significantly alter microparticle accumulation. Microparticle accumulation and anti-oxidant capacity of fresh frozen plasma was positively or negatively correlated, respectively, with the level of circulating microparticles in individual donors. The preparation protocol and the duration of storage in the freezer, independently and in combination, influenced the accumulation of microparticles in fresh frozen plasma units. In contrast, storage of thawed units for 24 h at 4 °C had no significant effect on the concentration of microparticles.

Force Sensing by the Vascular Protein Von Willebrand Factor is Tuned by a Strong Intermonomer Interaction

The large plasma glycoprotein von Willebrand factor (VWF) senses hydrodynamic forces in the blood stream and responds to elevated forces with abrupt elongation, thereby increasing its adhesiveness to platelets and collagen. Remarkably, forces on VWF are elevated at sites of vascular injury, where VWF's hemostatic potential is important to mediate platelet aggregation and to recruit platelets to the subendothelial layer. Adversely, elevated forces in stenosed vessels lead to an increased risk of VWF-mediated thrombosis. To dissect the remarkable force sensing ability of VWF, we have performed atomic force microscopy (AFM)-based single-molecule force measurements on dimers, the smallest repeating subunits of VWF multimers. We have identified a strong intermonomer interaction that involves the D4 domain and critically depends on the presence of divalent ions. Dissociation of this strong interaction occurred at forces above ∼50 pN and provided ∼80 nm of additional length to the elongation of dimers. Corroborated by the static conformation of VWF, visualized by AFM imaging, we estimate that under physiological conditions approximately half of VWF's constituent dimers are firmly closed via the strong intermonomer interaction. As firmly closed dimers drastically shorten VWF's effective length contributing to force sensing, they can be expected to markedly tune VWF's sensitivity to hydrodynamic flow in the blood and to thereby significantly affect VWF's function in hemostasis and thrombosis.

Force sensing by the vascular protein von Willebrand factor is tuned by a strong intermonomer interaction

The large plasma glycoprotein von Willebrand factor (VWF) senses hydrodynamic forces in the bloodstream and responds to elevated forces with abrupt elongation, thereby increasing its adhesiveness to platelets and collagen. Remarkably, forces on VWF are elevated at sites of vascular injury, where VWF's hemostatic potential is important to mediate platelet aggregation and to recruit platelets to the subendothelial layer. Adversely, elevated forces in stenosed vessels lead to an increased risk of VWF-mediated thrombosis. To dissect the remarkable force-sensing ability of VWF, we have performed atomic force microscopy (AFM)-based single-molecule force measurements on dimers, the smallest repeating subunits of VWF multimers. We have identified a strong intermonomer interaction that involves the D4 domain and critically depends on the presence of divalent ions, consistent with results from small-angle X-ray scattering (SAXS). Dissociation of this strong interaction occurred at forces above [Formula: see text]50 pN and provided [Formula: see text]80 nm of additional length to the elongation of dimers. Corroborated by the static conformation of VWF, visualized by AFM imaging, we estimate that in VWF multimers approximately one-half of the constituent dimers are firmly closed via the strong intermonomer interaction. As firmly closed dimers markedly shorten VWF's effective length contributing to force sensing, they can be expected to tune VWF's sensitivity to hydrodynamic flow in the blood and to thereby significantly affect VWF's function in hemostasis and thrombosis.

Emerging genetic and pharmacologic therapies for controlling hemostasis: beyond recombinant clotting factors.

For more than 3 decades, the scientific community has pursued gene correction of hemophilia, with the goal that an individual with congenitally deficient factor VIII or factor IX might synthesize adequate endogenous clotting factor to be relieved of burdensome repeated clotting factor infusions, as well as the emotional weight of continuous hemorrhage risk. Recent reports of successful factor IX gene therapy and partial correction of the bleeding phenotype have raised the bar for success for a robust crop of new clinical gene therapy efforts for both hemophilia A and B. At the same time that gene therapy is gaining momentum, suggesting the possibility of relief from regular intravenous coagulation protein replacement, a number of innovative technologies that enhance hemostatic potential independently of replacement factor administration are demonstrating success in human clinical application. Human clinical trial progress is reviewed regarding a recombinant bispecific IgG antibody to factors IXa and X that mimics factor VIII cofactor activity, as well as monoclonal antibody and short interfering RNA strategies that demonstrate hemostatic efficacy via opposing inhibitors of coagulation. These strategies, associated with prolonged hemostatic potential following subcutaneous (ACE910, ALN-AT3, Concizumab) or single administration (eg, gene therapy) make it possible to imagine a day when recombinant clotting factor administration, rather than being a daily preoccupation, is relegated to an adjunctive role in supporting more novel standard of care therapies.

Enhanced Global Hemostatic Potentials with a Bispecific Antibody to Factors IXa and X (ACE910) in the Whole Blood By Rotation Thromboelastometry (ROTEM)

Background: ACE910, a humanized bispecific antibody to factor (F) IXa and FX mimicking the functions of FVIII, exerts tenase activities without FVIII (a) (Kitazawa et al. Nature Medicine. 2012;18, 1570). In primate hemophilia A (HA) models, the hemostatic enhancing effect of ACE910 has been reported and the clinical study investigating the effect and safety of ACE910 for human HA patients is on-going. However, the hemostatic effect of ACE910 remains unquantified and difficult to be evaluated.Objectives: In this study, we evaluated the viscoelastmetric parameters in the whole blood obtained from HA patients under long-term treatment of ACE910 in phase 1 and its extension studies, utilizing rotation thromboelastometry (ROTEM), in order to investigate global hemostatic function of HA patients under treatment with ACE910.Methods: Ca2+-triggered hemostatic functions were assessed by ROTEM in the citrated whole blood samples obtained and stabilized at the indicated points from five severe HA cases with inhibitors (Case 1 : 39 BU/ml, 2 : 41 BU/ml, 3 : 17 BU/ml, 4 : 11 BU/ml and 5 : 111 BU/ml ) and two severe cases without inhibitors (Case 6 and 7) under subcutaneous administration of ACE910, with the different types of dosing regimen (group A with 0.3 mg/kg/week subcutaneous injection (loading dose of 1 mg/kg) for Case 1, 2, group B with 1 mg/kg/week (loading dose of 3 mg/kg) for Case 3, 4, group C with 3 mg/kg/week for Case 6 and group D changing from group A to group C for Case 5, 7) in a course of clinical study. The samples from the subjects prior to administration of ACE910 were spiked ex vivo with ACE910 and the hemostatic functions of them were also evaluated by ROTEM. The parameters of clot time (CT), clot formation time (CFT), maximum clot formation (MCF) and alpha angle were evaluated. The hemostatic potentials in the samples from twenty healthy volunteers and other ten HA patients with the various FVIII:C were evaluated by ROTEM as controls and the correlation between FVIII:C and CT was obtained. Annualized bleeding rates (ABR) of each case were calculated. The studies were approved by local ethics committee and the informed consent was obtained from each patient.Results: Addition of ACE910 (f.c. 10 or 30 μg/ml) into the sample from each case prior to administration of ACE910 shortened CT from 5,562 ± 374 sec (median 5,924 sec) to 1,475 ± 138 or 1,131 ± 82 sec, equivalent to FVIII:C 3.3 or 12.2 IU/dL, respectively, in an ACE910 concentration dependent manner. In group A, the plasma concentration of ACE910 got to 12 ± 5 μg/ml at 12 week. CT was shortened to 1,443 ± 24 sec, equivalent to FVIII:C 3.7 IU/dL maximally at 47 week consistent with the result of the spiked result. ABR decreased from 38.6 ± 25.8 to 1.1 ± 0.8, showing 97% decrease. As for Case 3 in group B (ACE910: 31 μg/ml at 12 week), CT was shortened to 1,164 sec, equivalent to FVIII:C 11.9 IU/dL maximally at 47 week consistent with the spiked data. ABR apparently decreased from 38.6 to 3.6. In Case 4 who dropped out the clinical study at 4 week, CT was shortened to 977 sec, equivalent to FVIII:C 22 IU/dL, maximally at 3 week, and continued to be shortened till 29 week (1,758 sec, equivalent to FVIII:C 1.1 IU/dL). In group C, the shortening of CT from 1,594 ± 103 sec, equivalent to FVIII:C 2.0 ± 1.0 (median 3.6) IU/dL (before administration of ACE910), to 1,226 ± 71 sec (8.5 ± 2.5 (median 10.4) IU/dL) (at steady state) was observed in consistent with the decrease of ABR from 8.1 to 1.1. In group D, CT was shortened from 3,405 ± 386 sec to 1,409 ± 85 sec, equivalent to FVIII:C 4.2 IU/dL after middle to high dose escalation in consistent with decrease of ABR from 22.3 ± 9.6 to 3.8 ± 2.6. Among all cases except Case 4, CT and ABR were of clinically relevant difference between before and after administration of ACE910.Conclusions: The comprehensive hemostatic function evaluated by ROTEM in hemophilia A patients irrespective of presence of inhibitors was improved after administration of ACE910 in a dose dependent fashion, resulting in the reduction of ABR, which suggested the hemostatic effectiveness of ACE910 for HA patients. DisclosuresYada:Chugai Pharmaceutical Co., Ltd: Research Funding. Nogami:Bayer, Novo Nordisk, Baxalta. Biogen: Research Funding; Chugai: Membership on an entity's Board of Directors or advisory committees; Bayer, NovoNordisk, Baxalta, Chugai, Kaketsuken, Pfizer, Biogen: Honoraria. Shida:Chugai Pharmacoceutical Co. Ltd.: Research Funding. Takeyama:Chugai Pharmaceutical Co., Ltd.: Research Funding. Kasai:Chugai Pharmaceutical Co., Ltd.: Employment. Shima:Chugai Pharmaceutical Co., Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biogen: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Baxalta: Honoraria, Research Funding; Novo Nordisk: Honoraria, Research Funding; Kaketsuken: Honoraria; Bayer: Honoraria, Research Funding.

Cold-Stored Platelets in PAS Exhibit Superior Hemostatic Potential

Background: Platelet transfusion is vital for hemostasis in patients with severe bleeding. Currently, platelets are stored at room temperature (RT) for 5 days with gentle agitation. However, this method results in a platelet storage lesion characterized by loss of platelet function and elevated risk of bacterial contamination. It has been shown that refrigeration of platelets at 4o C mitigates adverse effects and other logistical difficulties. Recently, FDA approved the use of cold-stored apheresis platelets for the resuscitation of actively bleeding patients for up to 3 days (5. 21 CFR 606.65(e) & 610.53(c)). We hypothesized that the refrigeration of platelets stored in 65% platelet additive solution (PAS), would better preserve the platelet contractile apparatus and overall hemostatic function. We measured clot stiffness, strength, crosslinking density and platelet contractile forces (PCF) using cone and plate rheometry; and quantified the microstructural architecture of the clots. To corroborate the rheological and morphological measurements with the clotting mechanism, we assayed thrombin generation, FXIII activity, clot retraction, and cytoskeletal rearrangement.Methods: Apheresis platelet (AP) concentrates collected from 4 healthy donors were isolated and stored in 65% Platelet additive solution (PAS) for 5 days at RT (22-24°C) or 4C (4°C). Clotting function was assayed after re-suspension in fresh frozen plasma (FFP). After initiating clotting with 20mM Ca2+, 100µL of the PRP sample was placed on the rheometer plate and clot stiffness was measured for 30 mins at 37o C at a small strain of 1Hz by tracking the evolving storage modulus (G'). Normal force measurements were made to quantify the force generated in the samples as a result of platelet contractile forces. Subsequently, a progressively increasing strain of 1 to 250% was applied until the clot was irreversibly deformed to estimate the yield point (clot strength). Scanning Electron Microscopy was performed on the clots from the rheological analysis to study the clot microstructure. Thrombin generation and FXIII activity were determined using Calibrated Automated Thrombogram and activity assays, respectively. Further, the cytoskeletal rearrangement was studied by activating the washed platelets with thrombin/ TRAP for 5, 10 and 15 seconds and estimating the G-actin to F-actin turnover by Western Blots. The results were analyzed using one-way ANOVA and a post hoc Tukey test for multiple comparisons. The groups were considered statistically significant for p values less than 0.05.Results: The rheological studies on clotting indicate that cold stored platelets form clots that have comparable properties to fresh platelets. RT-stored platelets formed weaker clots (Fig. 1A, B). These findings supported by the evidence of increased FXIII cross-linking activity (Fig.1C). Further, the normal force measurements indicate reduced force generation in RT-stored platelets which imply decreased platelet contractility (Fig. 1D). We also observed differences in geometry and orientation of individual fibrin fibers between fresh and stored platelets. Our preliminary results suggest that thrombin generation potential, crosslinking density, and platelet contractility may contribute to the mechanical strength and morphological differences between fresh and platelets stored at cold and room temperature.Conclusion: Cold-stored platelets form structurally and mechanically stable clots compared to 5 day stored RT platelets as demonstrated by the rheological and microstructural studies. Together, these data suggest that 4C platelets are hemostatically more active than the current standard-of-care and may offer a better solution to patients for resuscitation of bleeding trauma patients.Figure 1 Mechanical testing of evolving clot by rheometry. (A) RT-stored platelets form clots with decreased clot stiffness (G') compared to fresh (F) and cold-stored (4C) platelets (B) Clot strength was defined as the yield point where the clot deformed irreversibly. Clots formed from RT-stored platelets offer less resistance to strain; (C) Relative Cross Linking Density was estimated as the slope of stress-strain curve obtained from amplitude sweep. RT-stored platelet clots show less crosslinking; (D) less contractile force generation in RT platelets. * p <0.05, n=4. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The N-Linked Glycans of ADAMTS13 Are Critical Determinants of Conformation and Proteolytic Activity

Introduction: The multimeric size and haemostatic potential of Von Willebrand Factor (VWF) is regulated by the metalloproteinase ADAMTS13 that cleaves VWF within its A2 domain. Under conditions of shear stress, ADAMTS13 cleavage mediates VWF collagen and platelet binding function and therefore regulates the size of the growing thrombus. It has also recently been shown that an intra-domain interaction between the Spacer and CUB domains is present in ADAMST13 resulting in both "closed" and "open" ADAMTS13 molecules. A significant portion of the ADAMTS13 molecule is comprised of glycan structures with 10 N-linked glycosylation sites spaced throughout the molecule, while the exact functional role of these is not determined we hypothesised that they may affect ADAMTS13 conformation and proteolytic activity.Methods: Wild type and ADAMTS13 mutants lacking specific N-linked glycosylation sites were expressed in HEK293T cells as were ADAMTS13 variants truncated after the spacer domain (MDTCS), cysteine rich domain (MDTC) and the disintegrin-like domain (MD) and the isolated CUB domains. Terminal silaic acid residues and whole N-linked glycan chains were removed enzymatically with neuraminidase (As-AD13) and PNGase F (PNG-AD13) respectively. The ability of ADAMTS13 and its variants to cleave VWF was assessed under both static and flow conditions and binding between the spacer and CUB domains determined by immunoprecipitation based assays.Results: As-AD13 and PNG-AD13 both demonstrated significantly reduced cleavage of the small FRETS-76 substrate and full length VWF under static conditions and importantly under conditions of physiological shear stress. Removal of sialic acid or N-linked glycan chains from the MDTCS, MDTC and MD variants also reduced proteolysis, indicating that silylation of one or both of the N-linked glycans in the metalloproteinase domain is required for proper ADAMTS13 function. Neuraminidase digestion did not affect the binding of ADAMTS13 to VWF; however PNG-AD13 had significantly reduced binding to VWF but interestingly PNG-MDTCS bound VWF with normal affinity. Using co-immunoprecipitation we demonstrated altered interactions between the Spacer and CUB domains depending on their respective glycosylation status. We hypothesise that the N-linked glycans modulate the recently determined intra-molecular interaction within ADAMTS13, and their absence alters the conformation of ADAMTS13 in solution. To further investigate this we expressed ADAMTS13 mutants lacking specific N-linked glycosylation sites. Mutation of the conserved glycan sites in the CUB 1 (N1235) and CUB 2 (N1354) domains to glutamine did not affect the expression of ADAMTS13 but in contrast to PNG-AD13, both these mutants were gain-of-function, having significantly enhanced ability to interact with and proteolysis VWF under static and flow conditions. To assess the potential therapeutic use of these gain-of-function mutants and wild type ADAMTS13 in a thrombotic setting, we pre-formed thrombi on collagen surfaces under both anti and pro-coagulant conditions then perfused wild type or mutant ADAMTS13 over the thrombi surface and monitored the reduction in thrombi size. Surprisingly, neither wild type ADAMTS13 nor the N1235Q or N1354Q mutants were able to reduce the size of pre-formed thrombi even at high concentrations (200nM) and furthermore the presence of ADAMTS13 did not enhance the tissue plasminogen mediated reduction in fibrin rich thrombi.Conclusion: The N-linked glycans of ADAMTS13 have critical effects on ADAMTS13 function; with terminal sialic acid residues in the metalloproteinase domain being essential for proper proteolytic function and glycosylation of the Spacer and CUB domains required to modulate the open/closed conformation of ADAMTS13. In addition, our data also demonstrates that while ADAMTS13 is effective at controlling the size of the growing thrombus it is ineffective at cleaving VWF once a thrombus is formed and is thus unable to reduce the size of pre-formed thrombi. Together these data highlight the importance of N-linked glycosylation for proper ADAMTS13 function. DisclosuresNo relevant conflicts of interest to declare.

ACE910 Facilitates Its Hemostatic Effect with the Lower Concentration of Factor X Than That Required for Factor VIIa-Driven Coagulation

The hemostatic effect of bypassing agents such as recombinant (r) factor (F)VIIa and activated prothrombin complex concentrates (aPCC) for hemophilia A with inhibitors (HA-inh) is not always stable (Berntope, Haemophilia 2009). The mechanism(s) of its instability remain unclear, however. We have recently reported the HA-inh case showing the attenuated responsiveness to aPCC (Ogiwara, Int J Hematol. 2014). Some groups reported the hemostatic effects of the complex concentrates of FVIIa and FX (Shirahata, Haemophilia 2012) in HA-inh, suggesting that FX would play the key role in the hemostatic effect by FVIIa. ACE910, a humanized bispecific antibody to FIXa and FX mimicking the functions of FVIIIa, exerting FXase activities without FVIII(a) (Kitazawa, Nature Medicine 2012). In this study, we attempted to elucidate the dependency on FX of the FVIIa- and/or ACE910-driven coagulation. Firstly, the global hemostatic potentials in the whole blood samples obtained from the four HA-inh cases (Case 1, 2, 3 and 4) under perioperative hemostatic treatment with the intermittent administration of rFVIIa every 2-3hr were evaluated by Ca2+-triggered viscoelastometric assay with ROTEM. The first infusion of rFVIIa shortened CT (from 5,087 ± 1,261 to 1,157 ± 208 sec) and increased MCF (from 17 ± 8.7 to 58.8 ± 1.3 mm) in each case. Additional rFVIIa after the 7th administration in Case 1, the 13th in Case 2 and the 12th in Case 3 little affected CT and MCF as well as clinical symptom, indicative of poor responsiveness, while Case 4 showed the improvement of the parameters even after the frequent infusion of rFVIIa, identified as a responsive case. Thrombin generation (TG) triggered by TF (1pM) or TF (1pM) together with ellagic acid (0.3μM) was evaluated in the plasma from the cases with poor response. Peak thrombin (PeakTh) was little changed between pre- and post-additional infusion of rFVIIa in the cases with poor response, similar to the pattern of ROTEM. The level of FX antigen measured by an ELISA in the plasma was 90.5 ± 9.6 nM, showing 67% of normal control (~140 nM), of little difference among the four cases at the first administration of rFVIIa, while that in Case 1, 2 or 3 at the 7th, 13th or 12th administration, respectively, decreased to 39.1 ± 7.0 nM, equivalent to ~45% of that (86.8 ± 12.9 nM) kept in the responsive Case 4. Addition of FX (300nM) in the plasma of poor response to rFVIIa ex vivo increased PeakTh to ~80% of normal control, suggesting that FVIIa-driven hemostatic effect would be dependent upon FX. Furthermore, to investigate the FX-dependency of FVIIa- and ACE910-driven coagulation, TG in the reconstituted HA-inh model plasmas consisting of FX-deficient plasma in which FVIII was inactivated by an anti-FVIII polyclonal antibody (10BU/ml) with/without rFVIIa (50 and 150 nM) or ACE910 (10, 30 and 60 μg/ml) was evaluated in the presence of various concentrations of FX (f.c. 0 - 300 nM). The control experiment without rFVIIa or ACE910 showed the FX dose-dependent increase of PeakTh. In the plasmas with FX ranged from 50 to 300nM, PeakTh improved to almost normal level by rFVIIa as well as ACE910. Of note, with the lower concentration of FX (10-20 nM), PeakTh improved to almost normal level in the presence of ACE910, increased by 38 ± 2.4%, 45 ± 1.7% and 48 ± 0.8% compared to those in its absence, respectively, in an ACE910 dose-dependent manner, whilst the presence of rFVIIa little affected TG compared to those in its absence. Taken together, ACE910 could exert its hemostatic effect with the lower amount of FX than that required for the rFVIIa-driven coagulation. DisclosuresYada:Chugai Pharmaceutical Co., ltd: Research Funding. Nogami:Bayer, NovoNordisk, Baxalta, Chugai, Kaketsuken, Pfizer, Biogen: Honoraria; Bayer, Novo Nordisk, Baxalta. Biogen: Research Funding; Chugai: Membership on an entity's Board of Directors or advisory committees. Kitazawa:Chugai Pharmaceutical Co., Ltd: Employment, Equity Ownership, Patents & Royalties. Hattori:Chugai Pharmaceutical Co., Ltd: Employment, Equity Ownership, Patents & Royalties. Shima:Biogen: Honoraria, Research Funding; Bayer: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Baxalta: Honoraria, Research Funding; Novo Nordisk: Honoraria, Research Funding; Kaketsuken: Honoraria.

Comparison of hemostatic dressings for superficial wounds using a new spectrophotometric coagulation assay

BackgroundDue to demographical changes the number of elderly patients depending on oral anticoagulation is expected to rise. Prolonged bleeding times in case of traumatic injuries represent the drawback of these medications, not only in major trauma, but also in superficial wounds. Therefore, dressings capable of accelerating coagulation onset and shortening bleeding times are desirable for these patients.MethodsThe hemostatic potential and physical properties of different types of superficial wound dressings (standard wound pad, two alginates, chitosan, collagen (Lyostypt®), oxidized cellulose, and QuikClot®) were assessed in vitro. For this purpose the clotting times of blood under the influence of the named hemostatics from healthy volunteers were compared with Marcumar® or ASS® treated patients. For that, a newly developed coagulation assay based on spectrophotometric extinction measurements of thrombin activity was used.ResultsThe fastest coagulation onset was observed for oxidized cellulose (Ø 2.47 min), Lantor alginate-l (Ø 2.50 min) and QuikClot® (Ø 3.01 min). Chitosan (Ø 5.32 min) and the collagen Lyostypt® (Ø 7.59 min) induced clotting comparatively late. Regarding physical parameters, QuikClot® showed the lowest absorption capacity and speed while chitosan and both alginates achieved the highest. While oxidized cellulose displayed the best clotting times, unfortunately it also revealed low absorption capacity.ConclusionsAll tested specimens seem to induce clotting independently from the administered type of oral anticoagulant, providing the possibility to neglect the disadvantage in clotting times arising from anticoagulation on a local basis. QuikClot®, oxidized cellulose and unexpectedly alginate-l were superior to chitosan and Lyostypt®. Due to its additional well-known positive effect on wound healing alginate-l should be considered for further investigations.

Platelets and their role in cancer evolution and immune system.

Platelets are anucleate fragments formed from the cytoplasm of megakaryocytes and represent the smallest circulating hematopoietic cells. Thought for almost a century to possess solely hemostatic potentials, platelets actually play a much wider role in tissue regeneration and repair and interact intimately with tumor cells. On the one hand, tumor cells induce platelet aggregation, known to act as the trigger of cancer-associated thrombosis and on the other hand, platelets recruited to the tumor microenvironment interact directly with tumor cells favoring proliferation, and indirectly through the release of angiogenic and mitogenic proteins. Furthermore, platelets are immunosuppressive cells that protect metastatic cancer cells from surveillance by killer cells, nullifying the effects of immunotherapy.

Effects of recombinant human prothrombin on thrombin generation in plasma from patients with hemophilia A and B

The present study was carried out to investigate the impact of FII levels, and their increase, on the hemostatic potential in plasma from hemophilia A and B patients with and without inhibitors. Recombinant human factor (F) II (rhFII) was added ex vivo to plasma from 68 patients with hemophilia A and B, with or without inhibitors. The hemostatic potential as measured by thrombin generation (calibrated automated thrombogram [CAT]) was focused on the endogenous thrombin potential (ETP) as it has been shown to correlate with the clinical phenotype of bleeding in hemophilia patients and has also been used to guide bypassing therapy in hemophilia patients with inhibitors before elective surgery. The factor eight inhibitor bypassing agent (FEIBA(®) ) was used as a reference to the clinical situation. The study shows that rhFII concentration-dependently increased ETP by a similar magnitude in hemophilia A and B, both with and without inhibitors. Compared with FEIBA, rhFII showed a shallower concentration-response curve. In both types of hemophilia 100 mg L(-1) of rhFII roughly doubled the ETP. A corresponding response was obtained by 0.5 U mL(-1) of FEIBA. These data support the theory that FII is one of the major components responsible for the efficacy of FEIBA. The data also indicate that rhFII may be useful, alone or in combination with other coagulation factors, in some of the conditions for which FEIBA is used today, although more data are needed to substantiate this.

Persistent global hypercoagulability in long-term survivors of acute pulmonary embolism.

Hypercoagulable and/or hypofibrinolytic states are risk factors for venous thromboembolism (VTE) including acute pulmonary embolism. Current screening for thrombophilia is targeted towards identifying a specific defect and guidelines recommend a population-based rather than individualized strategy for anticoagulation treatment. We investigated whether there is a global hypercoagulable state in long-term survivors of pulmonary embolism no longer receiving therapeutic anticoagulation utilizing the overall haemostatic potential (OHP) assay, which assesses overall coagulation potential (OCP), OHP and overall fibrinolytic potential (OFP). Long-term survivors of acute pulmonary embolism were identified from a local registry and OHP assays were performed and compared with age and sex-matched controls without pulmonary embolism. Time courses of fibrin formation and degradation were measured by spectrophotometry (absorption 405 nm) after addition of tissue factor and tissue plasminogen activator to plasma. OHP assays were performed in 67 long-term survivors of single pulmonary embolism (7.9 ± 1.4 years after pulmonary embolism) and 20 age (61.7 ± 11.2 vs 56.6 ± 6.4 years, P = 0.06) and sex (P = 0.45)-matched controls. Survivors of pulmonary embolism were more hypercoagulable as reflected by significantly higher OCP (56.4 ± 13.0 vs 49.9 ± 6.9, P = 0.03) and had impaired fibrinolysis with higher OHP (12.6 ± 7.0 vs 5.9 ± 2.0, P < 0.001) and lower OFP (78.1 ± 9.4 vs 88.2 ± 2.9, P < 0.001) compared with controls. Importantly, these abnormalities in overall coagulation were independently predicted by levels of fibrinogen, platelet count, shortened activated partial thromboplastin time and inflammatory markers suggesting a multifactorial cause. Long-term survivors of pulmonary embolism demonstrate enhanced global coagulation and reduced fibrinolytic potential. Assessment of global coagulation may provide new insights into the aggregate effects of multiple prothombotic factors and long-term risk of VTE recurrence.

Hypercoagulable overall haemostatic potential is not intrinsically associated with PCOS but worsens with increasing BMI

Hypercoagulable overall haemostatic potential is not intrinsically associated with PCOS but worsens with increasing BMI