Thromboelastography in Children with Coagulation Factor Deficiencies.

Abstract The thromboelastograph produces a continuous profile of the rheological changes that occur during the process of coagulation using whole blood. This information can be transformed into a dynamic velocity profile of the changes in blood elasticity occurring during clotting. We used the TEG® hemostasis analyzer in patients with hemophilia A or B with and without inhibitors and other coagulation factor deficiencies (OFD), to study the thromboelastographic profiles in these patients. Materials and Methods: 62 children (6 months-19 years old) were enrolled according to IRB regulations. 29 children had severe hemophilia A (SHA), 4 moderate hemophilia A or B (Mod.H), 2 severe factor VII deficiency, 1 combined factor V and VIII deficiency, 1 VWD (type II B), 1 severe factor V deficiency, 1 Severe PAI deficiency, 19 normal controls (NC), and 4 SHA with inhibitors (SHA+I). All patients were studied 72 hours after the last dose of factor. Citrated whole blood was activated using recombinant human tissue factor (Innovin, Dade Behring Inc®) and recalcified using 0.2M CaCl2. In patients with central lines with heparin, a heparinase cup was used. The TEG® was run for ≥ 90 min. CBC with differential was obtained on all subjects. Results: There was no significant difference in the CBC parameters among patients. Analysis of the TEG data revealed the following: Table 1 TEG Parameters (mean values) SHA (n=29) Mod.H (n=4) SHA+I (n=4) OFD (n=6) Control(n=19) MTG:Max rate of thrombin generation; TMG: Time to MTG; R: Reaction Time; K: Time to reach an amplitude of 20mm; MA: Max. Amplitude MTG(mm*100/sec) 8.7 9.6 1.3 9 17 TMG(min) 27.5 16.6 62.7 17.5 8.9 R(min) 22 14 56 15 7 K(min) 7 4 41 4 2 Max.Amplitude, MA (mm) 59 56 12 58 62 The rate of thrombin generation as visualized by plotting the 1st derivative of the TEG course, in patients with SHA without inhibitors, showed that they could be divided into 2 groups based on MTG (</>9). When analysed the 2 groups showed the following characteristics (5 representative curves from each group are shown): Figure Figure Table 2 TEG Parameters (Mean values) MTG < 9 (n=16) MTG > 9 (n=13) p value TMA: Time to MA; MTG(mm*100/sec) 5.5 12.6 <0.001 TMG (min) 33 20 0.009 R(min) 26 16 0.004 K(min) 9 3.4 0.03 MA(mm) 56.1 62.3 0.01 TMA(min) 60 38 0.006 13/29 children with SHA had target joints and 69%of patients with target joints had a MTG<9. Conclusions: SHA patients have variable bleeding tendencies as seen by the variation in MTG. A lower MTG is associated with a higher incidence of target joints. This may provide a clue as to which patients may have the greatest benefit from primary prophylaxis. Patients with OFD have a TEG® profile similar to Mod.H patients. SHA+I have poor thrombin generation as seen by a significantly longer TMG and R time (p <0.05), compared to all subjects. The TEG may provide valuable clues to the severity of bleeding tendencies in patients with factor deficiencies. In additional observations (not shown), it appears that the TEG may be used to monitor the response to treatment with factor concentrates and tailor treatment with rFVIIa.

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Coinheritance of Factor V (FV) Leiden enhances thrombin formation and is associated with a mild bleeding phenotype in patients homozygous for the FVII 9726+5G>A (FVII Lazio) mutation

Blood ◽

10.1182/blood-2003-04-1199 ◽

2003 ◽

Vol 102 (12) ◽

pp. 4014-4020 ◽

Cited By ~ 32

Author(s):

Elisabetta Castoldi ◽

José W. P. Govers-Riemslag ◽

Mirko Pinotti ◽

Debora Bindini ◽

Guido Tans ◽

...

Keyword(s):

Thrombin Generation ◽

Clinical Phenotype ◽

Activated Protein C ◽

Coagulation Factor ◽

Factor Vii ◽

Factor V ◽

Factor X ◽

Fv Leiden ◽

Fvii Deficiency ◽

Thrombin Formation

Abstract We investigated the role of thrombophilic mutations as possible modifiers of the clinical phenotype in severe factor VII (FVII) deficiency. Among 7 patients homozygous for a cross-reacting material-negative (CRM-) FVII defect (9726+5G>A, FVII Lazio), the only asymptomatic individual carried FV Leiden. Differential modulation of FVII levels by intragenic polymorphisms was excluded by a FVII to factor X (FX) gene haplotype analysis. The coagulation efficiency in the FV Leiden carrier and a noncarrier was evaluated by measuring FXa, FVa, and thrombin generation after extrinsic activation of plasma in the absence and presence of activated protein C (APC). In both patients coagulation factor activation was much slower and resulted in significantly lower amounts of FXa and thrombin than in a normal control. However, more FXa and thrombin were formed in the plasma of the patient carrying FV Leiden than in the noncarrier, especially in the presence of APC. These results were confirmed in FV-FVII doubly deficient plasma reconstituted with purified normal FV or FV Leiden. The difference in thrombin generation between plasmas reconstituted with normal FV or FV Leiden gradually decreased at increasing FVII concentration. We conclude that coinheritance of FV Leiden increases thrombin formation and can improve the clinical phenotype in patients with severe FVII deficiency. (Blood. 2003;102:4014-4020)

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Hemophilia a Clots Generated with Recombinant Factor VIIa Differed in Structure and Composition from Those Formed with Factor VIII

Blood ◽

10.1182/blood.v128.22.3798.3798 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3798-3798

Author(s):

Lilley Leong ◽

Irina N. Chernysh ◽

Yifan Xu ◽

Cornell Mallari ◽

Billy Wong ◽

...

Keyword(s):

Factor Viii ◽

Whole Blood ◽

Neutralizing Antibodies ◽

Research Funding ◽

Thrombin Generation ◽

Hemophilia A ◽

Clot Formation ◽

Factor Vii ◽

Wild Type ◽

Independent Mechanism

Abstract Patients with severe factor VIII (FVIII) deficiency (hemophilia A [HemA]) develop neutralizing antibodies (inhibitors) against FVIII in up to ~30% of cases. For HemA patients with inhibitors, activated recombinant factor VII (rFVIIa) is a treatment option. High levels of rFVIIa are required for treating HemA patients with inhibitors to induce direct activation of factor X on the surface of activated platelets via a tissue factor (TF)-independent mechanism (Hoffman M, Monroe DM. Thromb Res. 2010;125(suppl 1):S16-S18). To assess how rFVIIa-mediated clot formation in HemA patients with inhibitors may differ from unaffected individuals, we compared the effect of rFVIIa on HemA versus control (or HemA supplemented with 100% FVIII) clot formation in human and/or mouse systems. By TF-induced thrombin generation assay, increasing rFVIIa from 5 nM to 100 nM did not appreciably alter the kinetics or extent of thrombin generation compared with the same human HemA plasma containing 100% FVIII. Confocal microscopy of human HemA plasma clots generated with 75 nM rFVIIa and TF showed few branching fibrin fibers and an open fibrin meshwork. In contrast, TF-induced coagulation of the same HemA plasma containing 100% FVIII formed fibrin clots with numerous branches, interconnecting to form a dense meshwork. To confirm that these findings reflect rFVIIa-mediated clot formation in vivo, we assessed the intrinsic coagulation of mouse HemA whole blood collected without anticoagulant and spiked with rFVIIa. Intrinsic coagulation with rFVIIa was assessed by T2 magnetic resonance (T2MR), a technique capable of monitoring the separation of whole blood into serum, loose-clot, and tight-clot compartments during coagulation (Skewis et al. Clin Chem. 2014;60:1174-1182; Cines et al. Blood. 2014;123:1596-1603). By T2MR, rFVIIa induced the separation of HemA whole blood into the serum and clot compartments, indicating that the reduced fibrin generation with rFVIIa did not interfere with whole blood coagulation. Furthermore, saphenous vein puncture of HemA mice treated with rFVIIa showed a dose-dependent decrease in clot times. Scanning electron microscopy of the clots extracted from these HemA mice indicated markedly different composition than clots extracted from wild-type mice. In wild-type clots, fibrin and polyhedral erythrocytes formed a large proportion of the total structures. In contrast, clots from rFVIIa-treated HemA mice consisted primarily of platelets and erythrocytes with forms intermediate between discoid and polyhedral but, surprisingly, low fibrin content. Taken together, these data suggest that rFVIIa-mediated clot formation may require greater activated platelet involvement, which would be consistent with the TF-independent mechanism of action proposed for rFVIIa in HemA. Finally, the compositional difference between clots from wild-type versus HemA mice dosed with rFVIIa suggest that evaluating HemA therapies for their ability to form more physiologic clots could be an approach to improve treatment options for patients with HemA. Disclosures Leong: Bayer: Employment. Xu:Bayer: Employment. Mallari:Bayer: Employment. Wong:Bayer: Employment. Sim:Bayer: Employment. Cuker:Stago: Consultancy; Genzyme: Consultancy; Amgen: Consultancy; Biogen-Idec: Consultancy, Research Funding; T2 Biosystems: Research Funding. Marturano:T2 Biosystems: Employment. Lowery:T2 Biosystems: Employment. Kauser:Bayer: Employment. Weisel:Bayer: Research Funding.

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Assessment of Bleeding Phenotype in Hemophilia Α By a Novel Point-of-Care Global Assay

Blood ◽

10.1182/blood-2019-131252 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4662-4662

Author(s):

Debnath Maji ◽

Michael A Suster ◽

Divyaswathi Citla Sridhar ◽

Maria Alejandra Pereda ◽

Janet Martin ◽

...

Keyword(s):

Whole Blood ◽

Research Funding ◽

Thrombin Generation ◽

Hemophilia A ◽

Point Of Care ◽

Blood Samples ◽

Healthy Group ◽

Significant Difference ◽

Bleeding Score ◽

Bleeding History

Introduction: Patients with Hemophilia A have considerable phenotypic heterogeneity with respect to clinical severity based on their baseline factor levels. As clinical bleeding risk is helpful to individualize factor replacement therapy in hemophilia patients, previous studies have utilized direct and indirect methods of thrombin generation to classify individual bleeding phenotypes, however, with variable results. An easy to use, point-of-care, global assay to assess bleed phenotype, can be a useful tool in the clinical setting to determine intensity of prophylaxis therapy for patients with hemophilia. We have previously introduced a novel, point-of-care (POC), dielectric microsensor, ClotChip, and demonstrated its sensitivity to factor replacement in patients with severe hemophilia A. We aim to further test the ability of ClotChip in assessment of a bleeding phenotype, as described by a bleeding score, in patients with hemophilia A. Methods: After IRB approval, 28 patients with hemophilia A of varying severity and well-characterized bleeding history, were enrolled in this study at the time of trough factor levels. The bleeding history was extracted from patient charts and included number of bleeds (joint and soft-tissue), annual factor usage in terms of units/kg, and number of target joints. These parameters were used to generate a bleeding score (range: 0 - 24), and patients were divided in to 2 categories with scores between 0 - 12 (n=14) and > 12 (n=14). Healthy volunteers (n=17) were accrued as controls. Whole blood samples were obtained by venipuncture into collection tubes containing 3.2% sodium citrate. Samples were then tested with the ClotChip within 2 hours of collection. ClotChip is based on the electrical technique of dielectric spectroscopy (DS) and features a low-cost (material cost < $1), small- sized (26mm × 9mm × 3mm), and disposable microfluidic biochip with miniscule sample volume (< 10 µL). The ClotChip readout was taken as the temporal variation in the real part of blood dielectric permittivity at 1 MHz. Our previous studies have shown that the ClotChip readout is sensitive to the global coagulation process and the time to reach a peak in permittivity (Tpeak) is a sensitive parameter to assess coagulation factor defects. Thrombin generation assay (TGA) using low tissue factor concentration was also performed on blood samples according to the manufacturer's direction. TGA was not available for 4 hemophilia and 2 control samples. Endogenous thrombin potential (ETP) parameter of TGA was used in this study to assess thrombin generation. Data are reported as mean ± standard deviation (SD). Analysis of variance (ANOVA) was used to test for statistical significance between groups with P < 0.05. Spearman's correlation test was used to derive correlation statistics. Results: ClotChip exhibited a mean Tpeak of 2186s ± 1560s for hemophilia patients in the group with higher bleeding scores (i.e. score >12), a mean Tpeak of 931s ± 496s for the group with lower bleeding scores (i.e. score <12) and a mean Tpeak of 441s ± 74s for the healthy group (Figure 1A). A significant difference in Tpeak was found between the group with higher bleeding scores compared to the group with lower bleeding scores (P = 0.002) as well as between higher bleeding scores and the healthy group (P < 0.0001). However, no significant difference in the TGA ETP parameter was detected between the groups with higher bleeding scores (mean ETP: 470 ± 814) and lower bleeding scores (mean ETP: 471 ± 897) (Figure 1B). ETP exhibited a statistical difference between the healthy group (mean ETP: 3462 ± 575) and both hemophilia groups (P < 0.0001). We also carried out studies to investigate the correlative power of the ClotChip Tpeak parameter to the TGA ETP parameter when including additional blood samples that were collected at various times during a hemophilia patient's prophylaxis regimen. The ClotChip Tpeak parameter exhibited strong negative correlation to the TGA ETP parameter (Spearman's rs= -0.73, P < 0.0001). Conclusions: Our studies suggest that a novel dielectric microsensor (ClotChip) could be useful in assessing bleeding phenotype in hemophilia A patients, allowing rapid assessment of hemostasis using a miniscule amount of whole blood (<10 µL) at the POC. Further studies are needed to determine if ClotChip data can be used to individualize prophylactic factor replacement regimens in hemophilia A patients. Disclosures Maji: XaTek, Inc: Patents & Royalties: 9,995,701. Suster:XaTek, Inc: Consultancy, Patents & Royalties: 9,995,701. Mohseni:XaTek, Inc: Consultancy, Patents & Royalties. Ahuja:XaTexk Inc.: Consultancy, Patents & Royalties, Research Funding; Rainbow Children's Foundation: Research Funding; Bayer: Consultancy; Biovertiv Sanofi: Consultancy; Genentech: Consultancy.

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Subcutaneous Marzeptacog Alfa (Activated) Versus Intravenous rFVIIa for Treatment of Episodic Bleeding in FVIII Deficient Hemophilia a Rats

Blood ◽

10.1182/blood-2021-151284 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3181-3181

Author(s):

Tom Knudsen ◽

Peter Johansen ◽

Jill Reckless ◽

Shraddha Desai ◽

Grant E. Blouse

Keyword(s):

Allometric Scaling ◽

Hemophilia A ◽

Treatment Success ◽

Coagulation Factor ◽

Factor Vii ◽

Success Rates ◽

Knock Out ◽

Exact Test ◽

Coagulation Factor Vii ◽

Significant Difference

Abstract Background: FVIII deficient knock-out (F8 -/-) rats mimic the bleeding incidents seen in severe human hemophilia A (HA). Subcutaneous (SQ) marzeptacog alfa activated (MarzAA), a novel, engineered recombinant activated coagulation Factor VII (rFVIIa) has been shown to effectively treat episodic bleeding in a pilot experiment in HA rats. This study evaluated the effect of single SQ doses of MarzAA and a single intravenous (IV) dose of rFVIIa on episodic bleeding in F8 -/- rats. Moreover, it compared the effect of SQ MarzAA and IV rFVIIa directly. Methods: Animals were allocated to treatment with either SQ vehicle, SQ MarzAA (60, 180 or 385 µg/kg) or IV rFVIIa (NovoSeven ®) (580 µg/kg) immediately after the bleeding was diagnosed. Doses were based on allometric scaling from humans (Nair AB and Jacob S. J Basic Clin Pharm 2016; 7: 27-31). The primary endpoint of the study was clinical efficacy as rated by a well-defined 4-point scale (Excellent, Good, Fair or Poor), and the efficacy assessment was either treatment success (Excellent or Good) or treatment failure (Fair or Poor) at the 24-hour timepoint. All personnel handling or assessing animals were blinded to the treatment status of each animal except those dosing animals who knew the route of administration. Results: A total of 86 F8 -/- rats was enrolled in the study. Of these, 61 rats presented treatment eligible bleeds between 3 and 10 weeks of age. No statistically significant difference in bleeding severity score were found across groups on diagnosis. As assessed by the clinical outcome at 24 hours, all three SQ MarzAA dose groups exhibited a statistically significant effect on treatment response when compared to SQ vehicle treatment (Figure 1). Conversely, no statistically significant effect could be identified when the single IV rFVIIa 580 µg/kg dose group was compared to vehicle. The overall treatment success rates at 24 hours were: SQ vehicle: 8%, SQ MarzAA 60 µg/kg: 58%, SQ MarzAA 180 µg/kg: 67%, SQ MarzAA 385 µg/kg: 85%, and IV rFVIIa 580 µg/kg: 33%. When compared directly using allometric scaling of clinical doses, SQ MarzAA at 385 µg/kg exhibited a statistically superior effect compared to IV rFVIIa at 580 µg/kg (p=0.0154, Fischer's exact test). Conclusion: Single doses of SQ MarzAA were effective in treating episodic bleeding in HA rats and statistically distinguishable from vehicle at all dose levels tested. When clinically relevant doses were compared directly to rFVIIa, SQ MarzAA compared favorably to IV rFVIIa. Taken together, these data provide robust nonclinical evidence that a single dose of SQ MarzAA may be successful in treating episodic bleeding when administered after bleeding has started. Figure 1 Figure 1. Disclosures Knudsen: Catalyst Biosciences: Current Employment, Current holder of individual stocks in a privately-held company. Blouse: Catalyst Biosciences: Current Employment.

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A mathematical model of flow-mediated coagulation identifies factor V as a modifier of thrombin generation in hemophilia A:

10.1101/495374 ◽

2018 ◽

Author(s):

Kathryn G Link ◽

Michael T Stobb ◽

Matthew G. Sorrells ◽

Maria Bortot ◽

Katherine Ruegg ◽

...

Keyword(s):

Mathematical Model ◽

Biological Networks ◽

Thrombin Generation ◽

Hemophilia A ◽

Factor Xa ◽

Coagulation Factor ◽

Experimental Models ◽

Unexpected Result ◽

Factor V ◽

Experimental Approaches

Hemophilia A is a bleeding disorder categorized as severe, mild, and moderate deficiencies in factor VIII (FVIII). Within these categories the variance in bleeding severity is significant and the origins unknown. The number of parameters that could modify bleeding are so numerous that experimental approaches are not feasible for considering all possible combinations. Consequently, we turn to a mathematical model of coagulation under flow to act as a screening tool to identify parameters that are most likely to enhance thrombin generation. We performed global sensitivity analysis on 110,000 simulations that varied coagulation factor levels by 50-150% of their normal values in humans while holding FVIII levels at 1%. These simulations identified low factor V (FV) levels as the strongest candidate, with additional enhancement when combined with high prothrombin levels. This prediction was confirmed in two experimental models: Partial FV inhibition boosted fibrin deposition in flow assays performed at 100 1/s on collagen-tissue factor surfaces using whole blood from individuals with mild and moderate FVIII deficiencies. Low FV (~50%) or partial FV inhibition also augmented thrombin generation in FVIII-inhibited or FVIII-deficient plasma in calibrated automated thrombography. These effects were amplified by high prothrombin levels in both experimental models. Our mathematical model suggests a mechanism in which FV and FVIII compete to bind to factor Xa to initiate thrombin generation in low FV, FVIII-deficient blood. This unexpected result was made possible by a mechanistic mathematical model, providing an example of the potential of such models in making predictions in complex biological networks.

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Inhibitors in Patients with Congenital Bleeding Disorders Other Than Hemophilia

Seminars in Thrombosis and Hemostasis ◽

10.1055/s-0037-1607441 ◽

2017 ◽

Vol 44 (06) ◽

pp. 595-603

Author(s):

Giuseppe Marano ◽

Carlo Mengoli ◽

Vanessa Piccinini ◽

Simonetta Pupella ◽

Stefania Vaglio ◽

...

Keyword(s):

Considerable Increase ◽

Hemophilia A ◽

Coagulation Factor ◽

Factor Ix ◽

Factor Vii ◽

Factor V ◽

Bleeding Disorders ◽

The Past ◽

Congenital Deficiency ◽

Coagulation Factor V

AbstractThe most worrying complication of replacement therapy for severe hemophilia A and B is currently the occurrence of inhibitory alloantibodies against infused factor VIII and factor IX, respectively. Inhibitors compromise the management of hemorrhage in affected patients, with a considerable increase in complications, disability, and costs. While these alloantibodies have been extensively studied in the past years in hemophilia A and B, those occurring in patients with other inherited bleeding disorders are less well characterized and still poorly understood, mostly due to the rarity of these hemorrhagic conditions. This narrative review will deal with inhibitors arising in patients with inherited bleeding disorders other than “classical” hemophilia, focusing in particular on those developing in patients with congenital deficiency of coagulation factor V, factor VII, factor XI, and factor XIII.

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Megakaryocyte/Platelet-Restricted FVIII Expression Using a Platelet Factor 4 Promoter: Determination of Activation State and In Vivo Efficacy during Thrombocytosis.

Blood ◽

10.1182/blood.v110.11.1142.1142 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1142-1142

Author(s):

Andrea L. Damon ◽

Jolyon Jesty ◽

Lesley E. Scudder ◽

Dmitri V. Gnatenko ◽

Wadie F. Bahou

Keyword(s):

Hemophilia A ◽

Storage Proteins ◽

Coagulation Factor ◽

Platelet Factor 4 ◽

P Value ◽

Factor V ◽

Assay Sensitivity ◽

Platelet Factor ◽

Altered Expression

Abstract Ectopic delivery of coagulation factor VIII (FVIII) to megakaryocytes (Mk) represents a viable approach for localized tenase generation by effectively concentrating the FVIIIa/FIXa enzyme-cofactor complex onto the negatively-charged phospholipid surface of activated platelets. While phenotypic correction has been demonstrated using hemophilia A (FVIII−/−) murine models in vivo, the activation state of platelet FVIII (pFVIII), optimal promoter choice, and phenotypic correction in the setting of a thrombocytotic stimulus remain unestablished. Preliminary microarray experiments using human platelets (N=5) demonstrated that the Mk-specific platelet factor 4 (PF4) transcripts were among the most abundant, prompting use of the 1.1 Kb PF4 promoter for Mk/platelet-restricted expression of human B-domain-deleted (hBDD) FVIII within the background of an exon 17-deleted mouse model of hemophilia A (PF4/hBDD/FVIII−/−). A chromogenic tenase assay using gel-filtered platelets from PF4/hBDD/FVIII−/− mice confirmed the presence of functional FVIII equivalent to 73 mU·1x109 platelets·mL−1 (N = 10 mice). In contrast, FVIII was not detectable in PF4/hBDD/FVIII−/− plasma (assay sensitivity <20 pM) or in platelets from FVIII−/− mice. The ectopic pFVIII did not affect the release and/or function of other a-granule storage proteins as established by parallel measurements of platelet factor V (FV) using a prothrombinase assay. Paired tenase assays (± thrombin) confirmed that pFVIII (unlike pFV) required thrombin cleavage for complete activation. To delineate the effects of a thrombocytotic stimulus on pFVIII expression and/or function, PF4/hBDD/FVIII−/− (N = 10) or FVIII−/− control mice (N = 5) were injected with thrombopoietin (TPO; 10μg/kg/day for 5 days) resulting in an 87% average increase in platelet count. Day 10 tenase assay of TPO-injected PF4/hBDD/FVIII−/− mice demonstrated a 66% reduction in pFVIII activity (25 mU FVIII·1x109 platelets·mL−1), unassociated with altered expression of a-granule-stored amyloid-b-precursor protein (AbPP) as a control for storage granule content; plasmatic FVIII remained undetectable. In contrast, the decrease in total platelet FVIII biomass was less pronounced in TPO-treated PF4/hBDD/FVIII−/− mice, representing a 35% reduction from 147.6 mU to 96 mU after TPO stimulation. The decreased pFVIII in TPO-stimulated PF4/hBDD/FVIII−/− mice correlated with loss of phenotypic correction as evaluated using tail bleeding survival rates: wild-type mice (100%; N= 5), FVIII−/− (0%; N=5), PF4/hBDD/FVIII−/− (TPO-naïve 60%; N=11), PF4/hBDD/FVIII−/− (TPO-treated 0%; N=7) (p value between PF4/hBDD/FVIII−/− mice with and without TPO stimulation = 0.002). While these data establish that Mk-directed pFVIII (unlike pFV) is proteolytically inactive upon platelet activation, they also imply that thrombocytotic stimuli negatively affect the pFVIII bioavailability and phenotypic efficacy. As importantly, the hemostatic efficacy of platelet FVIII correlates best with localized platelet FVIII delivery (FVIII concentration/platelet) and not the systemic platelet FVIII bioavailability.

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Ancylostoma caninum Anticoagulant Peptide Blocks Metastasis In Vivo and Inhibits Factor Xa Binding to Melanoma Cells In Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615117 ◽

1998 ◽

Vol 79 (05) ◽

pp. 1041-1047 ◽

Cited By ~ 20

Author(s):

Kathleen M. Donnelly ◽

Michael E. Bromberg ◽

Aaron Milstone ◽

Jennifer Madison McNiff ◽

Gordon Terwilliger ◽

...

Keyword(s):

Active Site ◽

Thrombin Generation ◽

Factor Xa ◽

Coagulation Factor ◽

Melanoma Cells ◽

Factor V ◽

Ancylostoma Caninum ◽

Metastatic Activity

SummaryWe evaluated the in vivo anti-metastatic activity of recombinant Ancylostoma caninum Anticoagulant Peptide (rAcAP), a potent (Ki = 265 pM) and specific active site inhibitor of human coagulation factor Xa originally isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with rAcAP (0.01-0.2 mg/mouse) prior to tail vein injection of LOX human melanoma cells resulted in a dose dependent reduction in pulmonary metastases. In order to elucidate potential mechanisms of rAcAP’s anti-metastatic activity, experiments were carried out to identify specific interactions between factor Xa and LOX. Binding of biotinylated factor Xa to LOX monolayers was both specific and saturable (Kd = 15 nM). Competition experiments using antibodies to previously identified factor Xa binding proteins, including factor V/Va, effector cell protease receptor-1, and tissue factor pathway inhibitor failed to implicate any of these molecules as significant binding sites for Factor Xa. Functional prothrombinase activity was also supported by LOX, with a half maximal rate of thrombin generation detected at a factor Xa concentration of 2.4 nM. Additional competition experiments using an excess of either rAcAP or active site blocked factor Xa (EGR-Xa) revealed that most of the total factor Xa binding to LOX is mediated via interaction with the enzyme’s active site, predicting that the vast majority of cell-associated factor Xa does not participate directly in thrombin generation. In addition to establishing two distinct mechanisms of factor Xa binding to melanoma, these data raise the possibility that rAcAP’s antimetastatic effect in vivo might involve novel non-coagulant pathways, perhaps via inhibition of active-site mediated interactions between factor Xa and tumor cells.

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AB1119 U9: A NOVEL CLINICALLY ORIENTED ULTRASONOGRAPHIC SCALE AS A USEFUL MARKER FOR MONITORING THERAPEUTIC RESPONSE IN RHEUMATOID ARTHRITIS (MULTI CENTERS STUDY)

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.2733 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 1848.2-1849

Author(s):

M. A. Mortada ◽

H. Eitta ◽

R. Elmallah ◽

A. Radwan ◽

A. Elsaman

Keyword(s):

Rheumatoid Arthritis ◽

Disease Activity ◽

Clinical Laboratory ◽

Pearson Correlation ◽

Response To Treatment ◽

P Value ◽

Clinical Parameters ◽

Biologic Dmards ◽

Das 28 ◽

Significant Difference

Background:Musculoskeletal Ultrasonography (MSUS) is now a widely used tool for monitoring of rheumatoid arthritis (RA). Although there are many proposed sets of composite scores, a fixed set of joints may not be an ideal tool to assess a disease like RA, which affects many joints and tendons in different presentations. In previous study (1) U9 score was proven to be correlated with disease activity parameters.Objectives:To determine whether US assessment using U9 score is useful for monitoring response to treatment for RA or not?Methods:A prospective, multicenter study were conducted in period from July 2019 to December 2019. All recruited RA patients were subjected to: Disease activity assessment by clinical disease activity indices (CDAI and DAS28 ESR). Functional status assessment by (HAQ) and ultrasonographic assessment using U9 score which include 8 joints (bilateral wrists,2ndMCP,3RDMCP and knees) plus most clinically affected joint or tendon (one joint or one tendon). Most clinically affected joints from 48 joints. Any affected tendons could be choosing. All targeted joints were evaluated according to EULAR guidlines and by EULAR/ OMERACT combined score (0-3). Targeted tendons were scored (0-3).All patients received their treatment (biologic and non biologic DMARDs) according to the decision of the treating physicians. No specific therapy is needed. CDAI and DAS28 ESR, HAQ and U9 score were repeated after 3 months to detect the response to change after receiving the therapy.Results:One hundred and forty patients (23.6% were male) with mean age 39.26±11.30 were recruited from 4 tertiary referral university hospitals.There was a significant difference (<0.001) between the first and second visits as regards clinical, laboratory and ultrasonographic parameters. DAS 28 decreased form (5.29±1.21) to (3.95±0.99), ESR decreased from (42.12±15.24) to (26.84±12.32), HAQ2 improved from (0.652±0.350) to (0.510±0.237) and U9 total US score decreased from (13.56±5.18) to (8.02±4.28).There was significant correlation between U9 ultrasonographic score and clinical parameters at both visits (table 1).Table 1.correlation between U9 ultrasonographic score and clinical parameters.U9 at 1stvisitU9 at 2ndvisitDAS-28Pearson Correlation(P value)0.806<0.0010.790<0.001CDAIPearson Correlation(P value)0.787<0.0010.773<0.001HAQPearson Correlation(P value)0.431<0.0010.317<0.001We found that the most suitable cut-off value of U9 score to predict high disease activity was 11.5 (sensitivity 85.7% and specificity 80.6%), cut off value for moderate disease activity was 5.5(sensitivity 83.2% and specificity 88%) and cut off value for low disease activity was 3.5 (sensitivity of 83.3% and specificity 57.1%). These results are summarized in the following table:Conclusion:U9 ultrasonographic score is very useful method for evaluating the monitoring the response of treatment.References:[1]Mortada, et al. Annals of the Rheumatic Diseases 2019;78:1009.Disclosure of Interests:None declared

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The Influence of Fibrinogen and Fibrin on Thrombin Generation - Evidence for Feedback Activation of the Clotting System by Clot Bound Thrombin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648947 ◽

1994 ◽

Vol 72 (05) ◽

pp. 713-721 ◽

Cited By ~ 88

Author(s):

Rachana Kumar ◽

Suzette Béguin ◽

H Coenraad Hemker

Keyword(s):

Snake Venom ◽

Thrombin Generation ◽

Platelet Rich Plasma ◽

Lag Time ◽

Factor Vii ◽

Factor V ◽

Clotting System ◽

Platelet Poor Plasma ◽

Normal Plasma ◽

System Factor

SummaryIn plasma the bulk of thrombin generation takes place after a clot has formed. We therefore investigated in what way the clot influences thrombin generation in plasma. The forming clot withdraws thrombin from free solution. Consequently less thrombin activity is found and less thrombin-inhibitor complexes are formed. The thrombin that is adsorbed to the clot reduces the lag time before thrombin generation in intrinsically or extrinsically triggered platelet poor plasma as well as in platelet rich plasma. We investigated the mechanism of this activation.Clots were obtained by recalcification of plasma or by the addition of thrombin-like enzymes (Reptilase, Agihal) from snake venoms. They were thoroughly washed until the washing fluid was devoid of any detectable clotting enzyme activity. In platelet poor plasma (PPP), thrombin-induced clots shorten the factor Va-dependent lag-time of thrombin generation in the extrinsic system as well as the factor VUIa-dependent thrombin generation in the intrinsic system. Factor V or factor VII preparations that in itself hardly influence thrombin generation patterns aquire the capacity to shorten these lag-times when incubated with clot. The last washing fluid of the clot is inactive. Snake venom induced clots are not active either. Clots that are incubated in heparinised plasma for 1 h or more are as active as clots from normal plasma are. A role of factor Xa can not be excluded but must be minor because a clot made by addition of thrombin to plasma from which the factors II, VII, IX and X have been removed is as active as a clot from normal plasma is.When added to recalcified platelet rich plasma (PRP), in which the lag-time of thrombin formation is dependent upon activation of platelet procoagulant phospholipid activity, any type of clot shortens the lagtime before the burst of thrombin generation. Clots that are obtained by snake venom enzymes are also active in this system. This indicates that fibrin alone is capable to induce the procoagulant phospholipid activity in platelets.We conclude that three known thrombin-dependent feedback activations in the clotting system (factor V, factor VIII and platelets) are efficiently supported by thrombin bound to the fibrin clot and that there is an additional activating effect of fibrin on the procoagulant action of platelets.

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