Role of factor VIII in clot growth and sensitivity to...
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C. Kluft 1 , S. Talens 1 , J Begieneman 1 , S. Naji 1 , N. Podoplelova 2 , F. Ataullakhanov 2 1 Good Biomarker Sciences, Leiden, Netherlands, www.GBSLeiden.com . 2 Dmitry Rogachev, National Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russia. OBJECTIVES METHODS Clot growth analysis was with the Thrombodynamics method (Hemacore). Inhibitors were from commercial sources. Citrated plasma was depleted of microvesicles, and corn trypsin inhibitor and excess (4µM) lipids were added before recalcification was used to start the process together with exposure of the tissue factor bound to the surface. The layer formation can be analyzed for several variables which include: • Thickness of the layer and • Rate of growth at each time point (notably at 2500 sec, Fig 4,5,6) www. gbsleiden.com C. Kluft: [email protected] S. Talens: [email protected] J. Begieneman: [email protected] S. Naji: [email protected] N. Podoplelova: [email protected] F. Ataullakhanov: [email protected] You are invited to visit GBS-Leiden for a demonstration of the clot growth analysis and testing of interesting clinical samples. • Reversible, direct factor Xa inhibitors inhibit only approximately half of clot growth (figure 5). – It involves inhibition of the contribution of factors XI, IX and VIII (see figure 3). • Inhibition is less potent with higher factor VIII (figure 6). The interaction of factor VIII with DXI inhibition is attributed to competition between the reversible factor Xa inhibitor and factor VIII (as substrate for factor Xa). It suggests that dosing of inhibitors be adjusted to factor VIII concentrations for decreased risk of bleeding or maintaining efficacy. Contact details RESULTS CONCLUSIONS Role of factor VIII in clot growth and sensitivity to reversible factor Xa inhibitors. Figure 3 : coagulation scheme: red is thrombin regulated process; green the tissue factor part Note: Polyphosphate is in sufficient amounts in plasma to drive the thrombin feed-back via exosite II of thrombin. Plasma clot growth is a new phenotype, recorded by video and characterized by clot sizes achieved in defined time periods and clot growth rate at each time point. We evaluated the role of factor VIII and the effects of reversible factor Xa inhibitors, rivaroxaban, apixaban and edoxaban. Figure 4 : Dose effect of reversible, direct factor Xa inhibitors (DXI) on clot size after 50 minutes. Final concentration DXI in haemophilia plasma on X-axis (FIX def plasma). Figure 5 : Dose effect of direct reversible factor Xa inhibitors on growth rate at 2500 seconds. Final concentration DXI in normal plasma on X-axis. The factor IX, X and XI participate all in final growth rate: initial growth is relatively intact with factor XI deficiency due to the role of TF (green in figure 3). Figure 2 : Dose effect of factor VIII (Advate) added to deficient plasma on clot growth. Figure 1 : Clot size followed in time for normal plasma and factor XI depleted and factor IX or VIII depleted plasma’s. 0 20 40 60 80 100 0 500 1000 1500 2000 2500 3000 3500 Clot size, um Time (min) Normal plasma FIX def (FVIII similar) FXI def Guide through the poster --Figure 1 shows reduced growth when factors XI, VIII or IX are absent. --Figure 2 shows correction by substitution of factor VIII in deficient plasma. Figure 3 shows the coagulation scheme: green is TF dependent; red is factor XI dependent. --Figure 4 shows that reversible factor Xa inhibitors do not inhibit growth and clot size very much in factor IX deficient plasma. --Figure 5 shows partial inhibition of the reversible factor Xa inhibitors in normal plasma on clot growth rate. Variation in factor VIII in plasma --Figure 6 shows inhibition by apixaban in plasma with different levels of factor VIII. Inhibition requires higher apixaban to reduce clot growth rate at higher factor VIII concentrations. Reversible, direct factor Xa inhibitors do not inhibit clot growth in factor IX deficient plasma (figure 4); and only partially in normal plasma (figure 5). 0 500 1000 1500 2000 2500 0 1000 2000 3000 4000 Clot size (µm) Time (sec) Advate in FVIII def plasma 0% FVIII 10% FVIII 15% FVIII 25% FVIII 50% FVIII 75% FVIII 100% FVIII Figure 6: Inhibition by apixaban in different concentrations (bars 0; 0.1; 0.2; 0.35 and 0.5 µM) of clot growth rate in plasma with different levels of factor VIII. With higher factor VIII, more apixaban is required to inhibit clot growth.
Transcript of Role of factor VIII in clot growth and sensitivity to...
C. Kluft1, S. Talens1, J Begieneman1, S. Naji1, N. Podoplelova2 , F. Ataullakhanov2
1 Good Biomarker Sciences, Leiden, Netherlands, www.GBSLeiden.com .2 Dmitry Rogachev, National Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russia.
OBJECTIVES METHODS
Clot growth analysis was with the Thrombodynamics method (Hemacore). Inhibitors were from commercial sources. Citrated plasma was depleted of microvesicles, and corn trypsin inhibitor and excess (4µM) lipids were added before recalcification was used to start the process together with exposure of the tissue factor bound to the surface. The layer formation can be analyzed for several variables which include: • Thickness of the layer and• Rate of growth at each time point (notably at 2500 sec, Fig 4,5,6)
www. gbsleiden.com
C. Kluft: [email protected]. Talens: [email protected]. Begieneman: [email protected]. Naji: [email protected]. Podoplelova: [email protected]. Ataullakhanov: [email protected]
You are invited to visit GBS-Leiden for a demonstration of the clot growth analysis and testing of interesting clinical samples.
• Reversible, direct factor Xa inhibitors inhibit only approximately half of clot growth (figure 5).– It involves inhibition of the contribution of factors XI, IX and VIII (see figure 3).
• Inhibition is less potent with higher factor VIII (figure 6).
The interaction of factor VIII with DXI inhibition is attributed to competition between the reversible factor Xa inhibitor and factor VIII (as substrate for factor Xa).
It suggests that dosing of inhibitors be adjusted to factor VIII concentrations for decreased risk of bleeding or maintaining efficacy.
Contact details
RESULTS
CONCLUSIONS
Role of factor VIII in clot growth and sensitivity to reversible factor Xa inhibitors.
Figure 3: coagulation scheme: red is thrombin regulated process; green the tissue factor part
Note: Polyphosphate is in sufficient amounts in plasma to drive the thrombin feed-back via exosite II of thrombin.
Plasma clot growth is a new phenotype, recorded by video and characterized by clot sizes achieved in defined time periods and clot growth rate at each time point.
We evaluated the role of factor VIII and the effects of reversible factor Xa inhibitors, rivaroxaban, apixaban and edoxaban.
Figure 4: Dose effect of reversible, direct factor Xa inhibitors (DXI) on clot size after 50 minutes.Final concentration DXI in haemophilia plasma on X-axis (FIX def plasma).
Figure 5: Dose effect of direct reversible factor Xa inhibitors on growth rate at 2500 seconds.Final concentration DXI in normal plasma on X-axis.
The factor IX, X and XI participate all in final growth rate: initial growth is relatively intact with factor XI deficiency due to the role of TF (green in figure 3).
Figure 2: Dose effect of factor VIII (Advate) added to deficient plasma on clot growth.
Figure 1: Clot size followed in time for normal plasma and factor XI depleted and factor IX or VIII depleted plasma’s.
0 20 40 60 80 1000
500
1000
1500
2000
2500
3000
3500
Clo
t siz
e, u
m
Time (min)
Normal plasma FIX def (FVIII similar) FXI def Guide through the poster
--Figure 1 shows reduced growth when factors XI, VIII or IX are absent.--Figure 2 shows correction by substitution of factor VIII in deficient plasma.
Figure 3 shows the coagulation scheme: green is TF dependent; red is factor XI dependent.
--Figure 4 shows that reversible factor Xa inhibitors do not inhibit growth and clot size very much in factor IX deficient plasma.
--Figure 5 shows partial inhibition of the reversible factor Xa inhibitors innormal plasma on clot growth rate.
Variation in factor VIII in plasma--Figure 6 shows inhibition by apixaban in plasma with different levels of
factor VIII. Inhibition requires higher apixaban to reduce clot growth rate athigher factor VIII concentrations.
Reversible, direct factor Xa inhibitors do not inhibit clot growth in factor IX deficient plasma (figure 4); and only partially in normal plasma (figure 5).
0
500
1000
1500
2000
2500
0 1000 2000 3000 4000
Clo
t siz
e (µ
m)
Time (sec)
Advate in FVIII def plasma
0% FVIII10% FVIII15% FVIII25% FVIII50% FVIII75% FVIII100% FVIII
Figure 6: Inhibition by apixaban in different concentrations (bars 0; 0.1; 0.2; 0.35 and 0.5 µM) of clot growth rate in plasma with different levels of factor VIII.
With higher factor VIII, more apixaban is required to inhibit clot growth.
