The glycoprotein Ib-IX-V complex contributes to tissue factor–independent thrombin generation by recombinant factor VIIa on the activated platelet surface

Blood ◽  
2008 ◽  
Vol 112 (8) ◽  
pp. 3227-3233 ◽  
Author(s):  
Cees Weeterings ◽  
Philip G. de Groot ◽  
Jelle Adelmeijer ◽  
Ton Lisman
Keyword(s):  
Tissue Factor ◽  
Cho Cells ◽  
Thrombin Generation ◽  
Recombinant Factor Viia ◽  
Factor Viia ◽  
Affinity Constant ◽  
Activate Factor ◽  
Platelet Surface ◽  
Static Conditions ◽  
Lines Of Evidence

Abstract Several lines of evidence suggest that recombinant factor VIIa (rFVIIa) is able to activate factor X on an activated platelet, in a tissue factor-independent manner. We hypothesized that, besides the anionic surface, a receptor on the activated platelet surface is involved in this process. Here, we showed that, in an ELISA setup, a purified extracellular fragment of GPIbα bound to immobilized rFVIIa. Surface plasmon resonance established a affinity constant (Kd) of approximately 20 nM for this interaction. In addition, CHO cells transfected with the GPIb-IX-V complex could adhere to immobilized rFVIIa, whereas wild-type CHO cells could not. Furthermore, platelets sti-mulated with a combination of collagen and thrombin adhered to immobilized rFVIIa under static conditions. Platelet adhesion was inhibited by treatment with O-sialoglycoprotein endopeptidase, which specifically cleaves GPIbα from the platelet surface. In addition, rFVIIa-mediated thrombin generation on the activated platelet surface was inhibited by cleaving GPIbα from its surface. In summary, 3 lines of evidence showed that rFVIIa interacts with the GPIb-IX-V complex, and this interaction enhanced tissue factor-independent thrombin generation mediated by rFVIIa on the activated platelet surface. The rFVIIa-GPIbα interaction could contribute to cessation of bleeding after administration of rFVIIa to patients with bleeding disorders.

Recombinant factor VIIa enhances deposition of platelets with congenital or acquired αIIbβ3 deficiency to endothelial cell matrix and collagen under conditions of flow via tissue factor–independent thrombin generation

Blood ◽  
2003 ◽  
Vol 101 (5) ◽  
pp. 1864-1870 ◽  
Author(s):  
Ton Lisman ◽  
Sultana Moschatsis ◽  
Jelle Adelmeijer ◽  
H. Karel Nieuwenhuis ◽  
Philip G. De Groot
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Recombinant Factor Viia ◽  
Factor Viia ◽  
Umbilical Vein ◽  
Novel Approach ◽  
Platelet Deposition ◽  
Platelet Aggregate ◽  
Umbilical Vein Endothelial Cells ◽  
Thrombin Formation

A novel approach to treat bleeding episodes in patients with Glanzmann thrombasthenia (GT) and perhaps also in patients receiving αIIbβ3 inhibitors is the administration of recombinant factor VIIa (rFVIIa). The mechanism of action of rFVIIa in these patients is, however, still unclear. We studied the effect of rFVIIa-mediated thrombin formation on adhesion of αIIbβ3-deficient platelets under flow conditions. Adhesion of αIIbβ3-deficient platelets to the extracellular matrix (ECM) of stimulated human umbilical vein endothelial cells or to collagen type III was studied using a model system with washed platelets and red cells. When αIIbβ3-deficient platelets were perfused over the surface at arterial shear rate for 5 minutes, a low surface coverage was observed (GT platelets, mean ± SEM, 37.5% ± 5.0%; normal platelets preincubated with an RGD-containing peptide, 7.4% ± 2.1%). When rFVIIa, together with factors X and II, was added to the perfusate, platelet deposition significantly increased (GT platelets, mean ± SEM, 67.0% ± 4.3%; normal platelets preincubated with an RGD-containing peptide, 48.2% ± 2.9%). The same effect was observed when normal platelets were pretreated with the commercially available anti-αIIbβ3 drugs abciximab, eptifibatide, or tirofiban. It was shown that tissue factor–independent thrombin generation (presumably induced by binding of rFVIIa to adhered platelets) was responsible for the increase in platelet deposition. In conclusion, defective adhesion of αIIbβ3-deficient platelets to ECM can be restored by tissue factor–independent rFVIIa-mediated thrombin formation. The enhanced generation of platelet procoagulant surface facilitates fibrin formation, so that lack of platelet aggregate formation might be compensated for.

scholarly journals A Systems Biology Model for the Coagulation Network in Non-Bleeding State Describes Baseline Activity of Coagulation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4238-4238
Author(s):  
Dooyoung Lee ◽  
Satyaprakash Nayak ◽  
Debra Pittman ◽  
Steven Arkin ◽  
Steven Martin ◽  
...  
Keyword(s):  
Healthy Volunteers ◽  
Prothrombin Time ◽  
Tissue Factor ◽  
Thrombin Generation ◽  
Recombinant Factor Viia ◽  
Factor Viia ◽  
Coagulation Activity ◽  
Low Level ◽  
Baseline Activity ◽  
Coagulation Pathway

Abstract Coagulation is a process crucial to stop bleeding from a damaged vessel. The network is comprised of a complex interplay of various pro-coagulant and anti-coagulant factors. Several systems models for the coagulation pathway have been published, as a way to investigate the pathway complexity. Most published models describe the thrombin generation profile (TGA) or prothrombin time (PT) following extrinsic pathway activation by tissue factor, or activated partial prothrombin time (aPTT) through activation of the intrinsic pathway by contact activation. In the clinical setting, thrombin-anti-thrombin complex (TAT) and prothrombin fragment 1+2 (PF1+2) are often used as biomarkers for in vivo coagulation activity in the non-bleeding state. Even in the absence of any treatment, there are detectable levels of PF1+2 and TAT in healthy volunteers indicating low level coagulation activity exists in the normal baseline, non-bleeding state. In this study, we developed a mathematical model for coagulation to describe the baseline activity of PF1+2 and TAT. We then used the model to understand the impact of the coagulation pathway activity during non-bleeding state on thrombin generation activated by tissue factor. First, a coagulation model from Hockin et al. (J Biol Chem. 2002;277(21):18322-33) was modified to describe internally generated data for thrombin generation and aPTT modulation following addition of various concentrations of recombinant factor VIIa or plasma derived factor Xa to normal or hemophilic human plasma. Next, protein synthesis and degradation were incorporated into the model and platelet-dependent reactions were tuned down to describe the baseline coagulation activity in non-bleeding healthy subjects. Using a simulated annealing algorithm, the new parameters were optimized to fit published data for PF1+2 and TAT in healthy volunteers and changes of PF1+2 following treatment with eptacog alfa (recombinant factor VIIa). In the model, the baseline coagulation activities can be described by a very low level of tissue factor, which is much lower than the detection limit of a regular ELISA method. Using this baseline model, we tested the effect of this baseline coagulation activity on tissue factor-activated thrombin generation, and found that very small baseline levels of activated enzymes significantly shorten the lag time of thrombin generation, but did not affect the peak thrombin. We also performed a global sensitivity analysis to identify key proteins in the coagulation network whose modulation will have the biggest impact on PF1+2 and TAT levels. Disclosures Lee: Pfizer Inc.: Employment. Nayak:Pfizer Inc.: Employment. Pittman:Pfizer Inc.: Employment. Arkin:Pfizer Inc.: Employment. Martin:Pfizer Inc.: Employment. Heatherington:Pfizer Inc.: Employment. Vicini:Pfizer Inc.: Employment. Hua:Pfizer Inc: Employment.

scholarly journals Factors IXa and Xa play distinct roles in tissue factor-dependent initiation of coagulation

Blood ◽  
1995 ◽  
Vol 86 (5) ◽  
pp. 1794-1801 ◽  
Author(s):  
M Hoffman ◽  
DM Monroe ◽  
JA Oliver ◽  
HR Roberts
Keyword(s):  
Tissue Factor ◽  
Platelet Activation ◽  
Thrombin Generation ◽  
Factor Viia ◽  
Factor Xa ◽  
Factor Ix ◽  
Main Role ◽  
Factor X ◽  
Platelet Surface ◽  
Factor Ixa

Tissue factor is the major initiator of coagulation. Both factor IX and factor X are activated by the complex of factor VIIa and tissue factor (VIIa/TF). The goal of this study was to determine the specific roles of factors IXa and Xa in initiating coagulation. We used a model system of in vitro coagulation initiated by VIIa/TF and that included unactivated platelets and plasma concentrations of factors II, V, VIII, IX, and X, tissue factor pathway inhibitor, and antithrombin III. In some cases, factor IX and/or factor X were activated by tissue factor- bearing monocytes, but in some experiments, picomolar concentrations of preactivated factor IX or factor X were used to initiate the reactions. Timed samples were assayed for both platelet activation and thrombin activity. Factor Xa was 10 times more potent than factor IXa in initiating platelet activation, but factor IXa was much more effective in promoting thrombin generation than was factor Xa. In the presence of VIIa/TF, factor X was required for both platelet activation and thrombin generation, while factor IX was only required for thrombin generation. We conclude that VIIa/TF-activated factors IXa and Xa have distinct physiologic roles. The main role of factor Xa that is initially activated by VIIa/TF is to activate platelets by generating an initial, small amount of thrombin in the vicinity of platelets. Factor IXa, on the other hand, enhances thrombin generation by providing factor Xa on the platelet surface, leading to prothrombinase formation. Only tiny amounts of factors IX and X need to be activated by VIIa/TF to perform these distinct functions. Our experiments show that initiation of coagulation is highly dependent on activation of small amounts of factors IXa and Xa in proximity to platelet surfaces and that these factors play distinct roles in subsequent events, leading to an explosion of thrombin generation. Furthermore, the specific roles of factors IXa and Xa generated by VIIa/TF are not necessarily reflected by the kinetics of factor IXa and Xa generation.

scholarly journals In vitro evidence of a tissue factor-independent mode of action of recombinant factor VIIa in hemophilia

Blood ◽  
2014 ◽  
Vol 124 (20) ◽  
pp. 3172-3174 ◽  
Author(s):  
Cecilia Augustsson ◽  
Egon Persson
Keyword(s):  
Tissue Factor ◽  
Mode Of Action ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Recombinant Factor Viia ◽  
Negative Impact ◽  
Factor Viia ◽  
Key Points ◽  
Independent Mode

Key Points The negative impact on thrombin generation of zymogen FVII competing with rFVIIa for TF is counteracted by FVII (auto)activation. Correction of hemophilia A occurs in a rFVIIa concentration range where detectable effects of FVII competition are minimal or absent.

Recombinant factor VIIa restores aggregation of αIIbβ3-deficient platelets via tissue factor–independent fibrin generation

Blood ◽  
2004 ◽  
Vol 103 (5) ◽  
pp. 1720-1727 ◽  
Author(s):  
Ton Lisman ◽  
Jelle Adelmeijer ◽  
Harry F. G. Heijnen ◽  
Philip G. de Groot
Keyword(s):  
Platelet Aggregation ◽  
Tissue Factor ◽  
Recombinant Factor Viia ◽  
Factor Viia ◽  
Microscopic Analysis ◽  
Coagulation Factors ◽  
Lag Phase ◽  
Electron Microscopic ◽  
Platelet Surface ◽  
Packed Structure

Abstract Recombinant factor VIIa (rFVIIa) is a safe and effective prohemostatic drug for patients with Glanzmann thrombasthenia (GT). However, the mechanism of action of rFVIIa in these patients is still unclear. Although patients with GT are characterized by a complete absence of platelet aggregation to a variety of agonists, it has been shown that GT platelets are able to form aggregates, provided polymerizing fibrin is present. We studied the effect of rFVIIa-mediated fibrin formation on aggregation of αIIbβ3-deficient platelets. When washed platelets from GT patients or platelets from healthy volunteers treated with an arginyl-glycyl-aspartyl–containing peptide were activated with collagen in the presence of rFVIIa and purified coagulation factors X, II, and fibrinogen, complete aggregation occurred after a lag phase. Fibrin generation proceeded via rFVIIa-mediated thrombin generation on the activated platelet surface independently of tissue factor. Electron microscopic analysis of αIIbβ3-independent platelet aggregates showed a densely packed structure suggestive of a true platelet-fibrin interaction and not via trapping of platelets into a fibrin network. Also, rFVIIa-mediated αIIbβ3-independent aggregation was demonstrated under conditions of flow using a collagen-coated surface. In conclusion, the efficacy of rFVIIa in GT patients might be explained by induction of αIIbβ3-independent platelet aggregation, which compensates the lack of αIIbβ3-dependent aggregation.

The Effect of Active Site-inhibited Factor VIIa on Tissue Factor-initiated Coagulation Using Platelets before and after Aspirin Administration

1997 ◽  
Vol 78 (04) ◽  
pp. 1202-1208 ◽  
Author(s):  
Marianne Kjalke ◽  
Julie A Oliver ◽  
Dougald M Monroe ◽  
Maureane Hoffman ◽  
Mirella Ezban ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Active Site ◽  
Large Scale ◽  
Thrombin Generation ◽  
Factor Viia ◽  
Dependent Manner ◽  
Antithrombotic Agent ◽  
Before And After ◽  
Ic50 Values

SummaryActive site-inactivated factor VIIa has potential as an antithrombotic agent. The effects of D-Phe-L-Phe-L-Arg-chloromethyl ketone-treated factor VIla (FFR-FVIIa) were evaluated in a cell-based system mimicking in vivo initiation of coagulation. FFR-FVIIa inhibited platelet activation (as measured by expression of P-selectin) and subsequent large-scale thrombin generation in a dose-dependent manner with IC50 values of 1.4 ± 0.8 nM (n = 8) and 0.9 ± 0.7 nM (n = 7), respectively. Kd for factor VIIa binding to monocytes ki for FFR-FVIIa competing with factor VIIa were similar (11.4 ± 0.8 pM and 10.6 ± 1.1 pM, respectively), showing that FFR-FVIIa binds to tissue factor in the tenase complex with the same affinity as factor VIIa. Using platelets from volunteers before and after ingestion of aspirin (1.3 g), there were no significant differences in the IC50 values of FFR-FVIIa [after aspirin ingestion, the IC50 values were 1.7 ± 0.9 nM (n = 8) for P-selectin expression, p = 0.37, and 1.4 ± 1.3 nM (n = 7) for thrombin generation, p = 0.38]. This shows that aspirin treatment of platelets does not influence the inhibition of tissue factor-initiated coagulation by FFR-FVIIa, probably because thrombin activation of platelets is not entirely dependent upon expression of thromboxane A2.

Impaired Platelet Function in Sept8-Deficient Mice In Vitro

2020 ◽  
Author(s):  
Kerstin Jurk ◽  
Katharina Neubauer ◽  
Victoria Petermann ◽  
Elena Kumm ◽  
Barbara Zieger
Keyword(s):  
Platelet Function ◽  
Thrombin Generation ◽  
Human Platelets ◽  
Platelet Surface ◽  
Integrin Αiibβ3 ◽  
Glycoprotein Vi ◽  
Fibrinogen Receptor ◽  
Static Conditions ◽  
The Impact

AbstractSeptins (Septs) are a widely expressed protein family of 13 mammalian members, recognized as a unique component of the cytoskeleton. In human platelets, we previously described that SEPT4 and SEPT8 are localized surrounding α-granules and move to the platelet surface after activation, indicating a possible role in platelet physiology. In this study, we investigated the impact of Sept8 on platelet function in vitro using Sept8-deficient mouse platelets. Deletion of Sept8 in mouse platelets caused a pronounced defect in activation of the fibrinogen receptor integrin αIIbβ3, α-granule exocytosis, and aggregation, especially in response to the glycoprotein VI agonist convulxin. In contrast, δ-granule and lysosome exocytosis of Sept8-deficient platelets was comparable to wild-type platelets. Sept8-deficient platelet binding to immobilized fibrinogen under static conditions was diminished and spreading delayed. The procoagulant activity of Sept8-deficient platelets was reduced in response to convulxin as determined by lactadherin binding. Also thrombin generation was decreased relative to controls. Thus, Sept8 is required for efficient integrin αIIbβ3 activation, α-granule release, platelet aggregation, and contributes to platelet-dependent thrombin generation. These results revealed Sept8 as a modulator of distinct platelet functions involved in primary and secondary hemostatic processes.

scholarly journals In Hemophilia Α Plasma Treated with Emicizumab, Factor IX Activation By Factor VIIa Drives Thrombin Generation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 17-17
Author(s):  
Dougald Monroe ◽  
Mirella Ezban ◽  
Maureane Hoffman
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Plasma Levels ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Factor Viia ◽  
Factor Ix ◽  
Factor X ◽  
Dose Dependent

Background.Recently a novel bifunctional antibody (emicizumab) that binds both factor IXa (FIXa) and factor X (FX) has been used to treat hemophilia A. Emicizumab has proven remarkably effective as a prophylactic treatment for hemophilia A; however there are patients that still experience bleeding. An approach to safely and effectively treating this bleeding in hemophilia A patients with inhibitors is recombinant factor VIIa (rFVIIa). When given at therapeutic levels, rFVIIa can enhance tissue factor (TF) dependent activation of FX as well as activating FX independently of TF. At therapeutic levels rFVIIa can also activate FIX. The goal of this study was to assess the role of the FIXa activated by rFVIIa when emicizumab is added to hemophilia A plasma. Methods. Thrombin generation assays were done in plasma using 100 µM lipid and 420 µM Z-Gly-Gly-Arg-AMC with or without emicizumab at 55 µg/mL which is the clinical steady state level. The reactions were initiated with low (1 pM) tissue factor (TF). rFVIIa was added at concentrations of 25-100 nM with 25 nM corresponding to the plasma levels achieved by a single clinical dose of 90 µg/mL. To study to the role of factor IX in the absence of factor VIII, it was necessary to create a double deficient plasma (factors VIII and IX deficient). This was done by taking antigen negative hemophilia B plasma and adding a neutralizing antibody to factor VIII (Haematologic Technologies, Essex Junction, VT, USA). Now varying concentrations of factor IX could be reconstituted into the plasma to give hemophilia A plasma. Results. As expected, in the double deficient plasma with low TF there was essentially no thrombin generation. Also as expected from previous studies, addition of rFVIIa to double deficient plasma gave a dose dependent increase in thrombin generation through activation of FX. Interestingly addition of plasma levels of FIX to the rFVIIa did not increase thrombin generation. Starting from double deficient plasma, as expected emicizumab did not increase thrombin generation since no factor IX was present. Also, in double deficient plasma with rFVIIa, emicizumab did not increase thrombin generation. But in double deficient plasma with FIX and rFVIIa, emicizumab significantly increased thrombin generation. The levels of thrombin generation increased in a dose dependent fashion with higher concentrations of rFVIIa giving higher levels of thrombin generation. Conclusion. Since addition of FIX to the double deficient plasma with rFVIIa did not increase thrombin generation, it suggests that rFVIIa activation of FX is the only source of the FXa needed for thrombin generation. So in the absence of factor VIII (or emicizumab) FIX activation does not contribute to thrombin generation. However, in the presence of emicizumab, while rFVIIa can still activate FX, FIXa formed by rFVIIa can complex with emicizumab to provide an additional source of FX activation. Thus rFVIIa activation of FIX explains the synergistic effect in thrombin generation observed when combining rFVIIa with emicizumab. The generation of FIXa at a site of injury is consistent with the safety profile observed in clinical use. Disclosures Monroe: Novo Nordisk:Research Funding.Ezban:Novo Nordisk:Current Employment.Hoffman:Novo Nordisk:Research Funding.

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