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Keywords:

  • platelet adhesion;
  • platelet aggregation;
  • recombinant factor VIIa;
  • thrombin;
  • thrombocytopenia

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Proteins
  6. Collagen- and fibrinogen-coated surfaces
  7. Blood collection
  8. Perfusion studies
  9. Determination of maximal thrombus height
  10. Intracellular calcium measurements
  11. Determination of phosphatidylserine-exposing platelets after perfusion
  12. Binding of FITC-labeled rFVIIa to adhered platelets under flow conditions
  13. Statistical analysis
  14. Results
  15. Thrombin generation via rFVIIa enhances platelet deposition to collagen under flow conditions at normal and reduced platelet count
  16. Recombinant FVIIa increases maximal thrombus height at normal and reduced platelet count
  17. Time course of platelet deposition to collagen at reduced platelet count in absence or presence of thrombin generation
  18. rFVIIa-mediated thrombin generation enhances calcium signaling and exposure of procoagulant phospholipids after adhesion to collagen at normal and reduced platelet count
  19. rFVIIa binds to collagen-adhered platelets in normal blood and in blood from severely thrombocytopenic patients
  20. Thrombin generation via rFVIIa increases platelet adhesion and platelet spreading to fibrinogen under flow conditions at normal and reduced platelet counts
  21. Discussion
  22. Acknowledgements
  23. References

Summary. Background: Recombinant factor VIIa (rFVIIa), which was developed for treatment of inhibitor-complicated hemophilia, appears suitable as prohemostatic agent in other clinical disorders including patients with thrombocytopenia. It is generally accepted that rFVIIa functions by enhancement of thrombin generation at the site of injury. It is, however, unknown if and how this affects platelet adhesion and aggregation. Objectives: To determine the effect of rFVIIa-mediated thrombin generation on platelet adhesion and aggregation under flow conditions at normal and reduced platelet counts. Methods: Washed platelets and red cells were combined to obtain plasma-free blood with different platelet counts. The reconstituted blood was perfused over a collagen- or fibrinogen-coated surface in the absence or presence of a thrombin generating system consisting of purified coagulation factors rFVIIa, factor (F)X and prothrombin. Results: Addition of coagulation factors rFVIIa, FX and prothrombin to washed platelets and red cells enhanced platelet adhesion and aggregation to collagen and adhesion and spreading to fibrinogen at normal platelet count and at platelet numbers as low as 10 000 µL−1. rFVIIa-mediated thrombin generation enhanced the activation state of platelets as measured by intracellular calcium fluxes, and enhanced the exposure of procoagulant phospholipids as measured by annexin A5 binding. Conclusions: Taken together, increased platelet adhesion and aggregation by rFVIIa-mediated thrombin formation may explain the therapeutic effects of rFVIIa in thrombocytopenic conditions and in patients with a normal platelet count by (i) enhancement of primary hemostasis and (ii) enhancement of procoagulant surface leading to elevated fibrin formation.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Proteins
  6. Collagen- and fibrinogen-coated surfaces
  7. Blood collection
  8. Perfusion studies
  9. Determination of maximal thrombus height
  10. Intracellular calcium measurements
  11. Determination of phosphatidylserine-exposing platelets after perfusion
  12. Binding of FITC-labeled rFVIIa to adhered platelets under flow conditions
  13. Statistical analysis
  14. Results
  15. Thrombin generation via rFVIIa enhances platelet deposition to collagen under flow conditions at normal and reduced platelet count
  16. Recombinant FVIIa increases maximal thrombus height at normal and reduced platelet count
  17. Time course of platelet deposition to collagen at reduced platelet count in absence or presence of thrombin generation
  18. rFVIIa-mediated thrombin generation enhances calcium signaling and exposure of procoagulant phospholipids after adhesion to collagen at normal and reduced platelet count
  19. rFVIIa binds to collagen-adhered platelets in normal blood and in blood from severely thrombocytopenic patients
  20. Thrombin generation via rFVIIa increases platelet adhesion and platelet spreading to fibrinogen under flow conditions at normal and reduced platelet counts
  21. Discussion
  22. Acknowledgements
  23. References

Recombinant factor VIIa (rFVIIa, NovoSeven®; Novo Nordisk A/S, Bagsværd, Denmark) represents a recent advancement in the treatment of inhibitor-complicated hemophilia [1]. Moreover, increasing clinical evidence suggests that rFVIIa is a safe and effective hemostatic drug for patients with different types of coagulopathies, including patients with liver disease [2,3], platelet disorders including von Willebrand's disease [4–6], and patients treated with antithrombotic drugs [7,8]. Also, rFVIIa appears to be effective in patients without coagulation disorders who are bleeding as a result of extensive surgery or major trauma [9,10]. The use of rFVIIa in a number of these novel indications is currently being tested in clinical trials [11,12]. Furthermore, rFVIIa has recently been registered in Europe for use in patients with FVII deficiency and patients with Glanzmann thrombasthenia (GT) refractory to platelet transfusion.

Several small human studies and one rabbit study suggest that rFVIIa is a safe and effective prohemostatic agent in patients with thrombocytopenia [13–15]. Currently, a controlled study investigating the use of rFVIIa in thrombocytopenic patients with moderate to severe bleeds after allogeneic stem cell transplantation is in progress [12].

Although convincing clinical evidence for the efficacy of rFVIIa in its various applications is rapidly accumulating, relatively little is known about its mechanism of action (reviewed in [16]). It is generally assumed that rFVIIa enhances thrombin generation at the site of vascular damage. In vitro experiments showed that enhancement of thrombin generation by rFVIIa can proceed via tissue factor (TF)-dependent [17–19] or -independent [20–22] pathways, and it has been postulated that both mechanisms are operative in vivo[23]. By which mechanisms enhancement of thrombin generation by rFVIIa ultimately leads to induction of hemostasis is uncertain, but this may include enhancement of fibrin formation [18] and platelet activation [24], protection of the fibrin clot against premature fibrinolysis by means of enhanced or accelerated activation of thrombin activatable fibrinolysis inhibitor [18], or by changes in fibrin structure [25].

It has been postulated that the mechanism of action of rFVIIa in thrombocytopenia involves TF-independent enhancement of thrombin generation on activated platelets [26]. Evidence comes from the observation that rFVIIa results in faster platelet activation and faster initial thrombin generation under thrombocytopenic conditions in a model study employing purified platelets and coagulation factors [27], and in a platelet-rich plasma model [28]. In these models, however, thrombin generation did not normalize on addition of rFVIIa. Also, the effect of rFVIIa-mediated enhancement of thrombin generation on platelet functions has not been considered in these experiments. Whole blood studies showed that rFVIIa, under conditions of thrombocytopenia, enhances fibrin deposition under flow conditions, but has no effect on platelet adhesion or aggregation [29]. It might, however, be possible that the fibrin generated in this model competes with platelets for binding to the surface, and thereby masks part of the platelet-promoting effect of rFVIIa.

Recently, we reported that thrombin generation mediated by rFVIIa enhanced deposition of αIIbβ3-deficient platelets to collagen and to the extracellular matrix of cultured human umbilical vein endothelial cells under flow conditions [22]. We speculated that enhanced deposition of αIIbβ3-deficient platelets might compensate for the lack of aggregation by facilitating fibrin formation due to increased available procoagulant platelet surface. This enhanced fibrin formation might cause platelet aggregation independent of αIIbβ3, which could also contribute to induction of hemostasis in patients with GT [30].

In this study, we investigated the effect of rFVIIa-mediated thrombin generation on platelet deposition to collagen under flow conditions at normal and reduced platelet count using the reconstituted blood model as described previously to investigate whether enhancement of platelet deposition is a general consequence of enhancement of thrombin generation via rFVIIa.

Proteins

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Proteins
  6. Collagen- and fibrinogen-coated surfaces
  7. Blood collection
  8. Perfusion studies
  9. Determination of maximal thrombus height
  10. Intracellular calcium measurements
  11. Determination of phosphatidylserine-exposing platelets after perfusion
  12. Binding of FITC-labeled rFVIIa to adhered platelets under flow conditions
  13. Statistical analysis
  14. Results
  15. Thrombin generation via rFVIIa enhances platelet deposition to collagen under flow conditions at normal and reduced platelet count
  16. Recombinant FVIIa increases maximal thrombus height at normal and reduced platelet count
  17. Time course of platelet deposition to collagen at reduced platelet count in absence or presence of thrombin generation
  18. rFVIIa-mediated thrombin generation enhances calcium signaling and exposure of procoagulant phospholipids after adhesion to collagen at normal and reduced platelet count
  19. rFVIIa binds to collagen-adhered platelets in normal blood and in blood from severely thrombocytopenic patients
  20. Thrombin generation via rFVIIa increases platelet adhesion and platelet spreading to fibrinogen under flow conditions at normal and reduced platelet counts
  21. Discussion
  22. Acknowledgements
  23. References

rFVIIa was a generous gift from R. Røjkjær (Novo Nordisk, Måløv, Denmark). FITC-labeled FVIIa was prepared by incubating rFVIIa for 30 min at 37 °C with a 25-fold molar excess of the FITC-labeled active site inhibitor Phe-Pro-Arg-chloromethylketone (purchased from U.S. Biological, Swampscott, MA, USA), and subsequent separation of free label by Q-Sepharose chromatography and dialysis as described [31]. Factor (F)X was purified from fresh-frozen plasma by immunoaffinity chromatography followed by Q-Sepharose chromatography as previously described [32]. Prothrombin was purified from freshly frozen plasma according to Koedam et al. [33]. Collagen type III was from Sigma (St Louis, MO, USA). FITC-labeled annexin A5 was from Nexins Research (Hoeven, the Netherlands).

Collagen- and fibrinogen-coated surfaces

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Proteins
  6. Collagen- and fibrinogen-coated surfaces
  7. Blood collection
  8. Perfusion studies
  9. Determination of maximal thrombus height
  10. Intracellular calcium measurements
  11. Determination of phosphatidylserine-exposing platelets after perfusion
  12. Binding of FITC-labeled rFVIIa to adhered platelets under flow conditions
  13. Statistical analysis
  14. Results
  15. Thrombin generation via rFVIIa enhances platelet deposition to collagen under flow conditions at normal and reduced platelet count
  16. Recombinant FVIIa increases maximal thrombus height at normal and reduced platelet count
  17. Time course of platelet deposition to collagen at reduced platelet count in absence or presence of thrombin generation
  18. rFVIIa-mediated thrombin generation enhances calcium signaling and exposure of procoagulant phospholipids after adhesion to collagen at normal and reduced platelet count
  19. rFVIIa binds to collagen-adhered platelets in normal blood and in blood from severely thrombocytopenic patients
  20. Thrombin generation via rFVIIa increases platelet adhesion and platelet spreading to fibrinogen under flow conditions at normal and reduced platelet counts
  21. Discussion
  22. Acknowledgements
  23. References

Collagen type III was solubilized in 50 mm acetic acid and sprayed on Thermanox (Nunc, Naperville, IL, USA) or glass coverslips [the latter were used for (immuno)fluorescence studies] using a retouching airbrush (Badger model 100; Badger Brush, Franklin Park, IL, USA) at a density of 30 µg cm−2. Fibrinogen was immobilized onto Thermanox coverslips by a coating procedure (100 µg mL−1, 1 h at room temperature). After the spraying or coating procedure, coverslips were blocked for 1 h at room temperature with 1% human albumin in phosphate-buffered saline (PBS).

Blood collection

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Proteins
  6. Collagen- and fibrinogen-coated surfaces
  7. Blood collection
  8. Perfusion studies
  9. Determination of maximal thrombus height
  10. Intracellular calcium measurements
  11. Determination of phosphatidylserine-exposing platelets after perfusion
  12. Binding of FITC-labeled rFVIIa to adhered platelets under flow conditions
  13. Statistical analysis
  14. Results
  15. Thrombin generation via rFVIIa enhances platelet deposition to collagen under flow conditions at normal and reduced platelet count
  16. Recombinant FVIIa increases maximal thrombus height at normal and reduced platelet count
  17. Time course of platelet deposition to collagen at reduced platelet count in absence or presence of thrombin generation
  18. rFVIIa-mediated thrombin generation enhances calcium signaling and exposure of procoagulant phospholipids after adhesion to collagen at normal and reduced platelet count
  19. rFVIIa binds to collagen-adhered platelets in normal blood and in blood from severely thrombocytopenic patients
  20. Thrombin generation via rFVIIa increases platelet adhesion and platelet spreading to fibrinogen under flow conditions at normal and reduced platelet counts
  21. Discussion
  22. Acknowledgements
  23. References

Blood was drawn from healthy volunteers who denied ingestion of aspirin or other non-steroidal anti-inflammatory drugs (NSAIDs) for the preceding 10 days into one-tenth volume 3.4% sodium citrate.

In selected experiments, blood from thrombocytic patients with myeloproliferative disorders receiving chemotherapy was used. The platelet counts in these patients were 47 000, 13 000, 22 000, 15 000 and 9000 per µL, respectively. Patient blood was obtained with full informed consent with approval by the local Medical Ethics Committee.

Perfusion studies

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Proteins
  6. Collagen- and fibrinogen-coated surfaces
  7. Blood collection
  8. Perfusion studies
  9. Determination of maximal thrombus height
  10. Intracellular calcium measurements
  11. Determination of phosphatidylserine-exposing platelets after perfusion
  12. Binding of FITC-labeled rFVIIa to adhered platelets under flow conditions
  13. Statistical analysis
  14. Results
  15. Thrombin generation via rFVIIa enhances platelet deposition to collagen under flow conditions at normal and reduced platelet count
  16. Recombinant FVIIa increases maximal thrombus height at normal and reduced platelet count
  17. Time course of platelet deposition to collagen at reduced platelet count in absence or presence of thrombin generation
  18. rFVIIa-mediated thrombin generation enhances calcium signaling and exposure of procoagulant phospholipids after adhesion to collagen at normal and reduced platelet count
  19. rFVIIa binds to collagen-adhered platelets in normal blood and in blood from severely thrombocytopenic patients
  20. Thrombin generation via rFVIIa increases platelet adhesion and platelet spreading to fibrinogen under flow conditions at normal and reduced platelet counts
  21. Discussion
  22. Acknowledgements
  23. References

Perfusions were carried out in a single-pass perfusion chamber as described previously [34]. Perfusions were carried out with reconstituted blood, which was prepared as follows. Platelet-rich plasma (PRP) was prepared from whole blood by centrifugation (10 min at 200 × g at room temperature). The PRP was acidified by addition of one-tenth volume of ACD (2.5% trisodium citrate, 1.5% citric acid, and 2% D-glucose), and the platelets were spun down (500 × g, 15 min). The platelet pellet was resuspended in HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid)–Tyrode buffer (10 mm HEPES, 137 mm NaCl, 2.68 mm KCl, 0.42 mm NaH2PO4, 1.7 mm MgCl2, 5 mm D-glucose, pH 6.5). Prostacyclin (PGI2, 10 ng mL−1) was added to prevent platelet activation during the subsequent washing step. Platelets were spun down and resuspended in a small volume of HEPES–Tyrode buffer. The platelets were diluted in human albumin solution (HAS; 4% human albumin, 4 mm KCl, 124 mm NaCl, 20 mm NaHCO3, 2 mm Na2SO4, 1.5 mm MgCl2, 5 mm D-glucose, pH 7.35).

Red cells were washed twice with 0.9% NaCl containing 5 mm D-glucose (2000 × g, 5 min), and finally cells were packed (2000 × g, 15 min).

Different amounts of platelets were mixed with red cells to obtain reconstituted blood with a hematocrit of 40% and platelet counts of 200 000, 100 000, 50 000, 25 000 and 10 000 per µL. The reconstituted blood was preincubated with buffer or a mixture of clotting factors [rFVIIa (1.2 µg mL−1), FX (10 µg mL−1), and prothrombin (20 ng mL−1)] in the presence of calcium chloride (3 mm) for 5 min at 37 °C and perfused for 5 min at a shear rate of 1600 s−1. After perfusion, slides were washed with HEPES buffer (10 mm HEPES, 150 mm NaCl, pH 7.35) and fixed in 0.5% glutaraldehyde in PBS. Subsequently, slides were dehydrated in methanol and stained with May–Grünwald and Giemsa as described previously [35]. Platelet adhesion was evaluated using computer-assisted analysis with OPTIMAS 6.0 software [Dutch Vision Systems (DVS), Breda, the Netherlands], and was expressed as the percentage of the surface covered with platelets.

Determination of maximal thrombus height

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Proteins
  6. Collagen- and fibrinogen-coated surfaces
  7. Blood collection
  8. Perfusion studies
  9. Determination of maximal thrombus height
  10. Intracellular calcium measurements
  11. Determination of phosphatidylserine-exposing platelets after perfusion
  12. Binding of FITC-labeled rFVIIa to adhered platelets under flow conditions
  13. Statistical analysis
  14. Results
  15. Thrombin generation via rFVIIa enhances platelet deposition to collagen under flow conditions at normal and reduced platelet count
  16. Recombinant FVIIa increases maximal thrombus height at normal and reduced platelet count
  17. Time course of platelet deposition to collagen at reduced platelet count in absence or presence of thrombin generation
  18. rFVIIa-mediated thrombin generation enhances calcium signaling and exposure of procoagulant phospholipids after adhesion to collagen at normal and reduced platelet count
  19. rFVIIa binds to collagen-adhered platelets in normal blood and in blood from severely thrombocytopenic patients
  20. Thrombin generation via rFVIIa increases platelet adhesion and platelet spreading to fibrinogen under flow conditions at normal and reduced platelet counts
  21. Discussion
  22. Acknowledgements
  23. References

The maximal height of thrombi obtained by perfusion over a collagen-coated surface was determined using confocal laser scanning microscopy as described previously [36]. Perfusions were performed over collagen coated to a glass coverslip. After perfusion, coverslips were fixed in 3% paraformaldehyde/0.025% glutaraldehyde in PBS. Coverslips were blocked with 1% bovine serum albumin (BSA) in PBS for 30 min, and permeabilized with 0.5% Triton X-100 in PBS for 5 min. The coverslips were subsequently incubated with 1 µg mL−1 TRITC-labeled phalloidin (Molecular Probes, Eugene, OR, USA) in 1% BSA/PBS to stain the cytoskeleton. Coverslips were mounted in VECTASHIELD mounting medium (Vector Laboratories, Burlingame, CA, USA) on concave slides with a depth of 0.79 mm. The maximal thrombus height was measured with a confocal laser scanning microscope (Leica TCS 4D, Heidelberg, Germany). For each slide, 10 fields of 0.25 × 0.25 mm2 were measured.

Intracellular calcium measurements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Proteins
  6. Collagen- and fibrinogen-coated surfaces
  7. Blood collection
  8. Perfusion studies
  9. Determination of maximal thrombus height
  10. Intracellular calcium measurements
  11. Determination of phosphatidylserine-exposing platelets after perfusion
  12. Binding of FITC-labeled rFVIIa to adhered platelets under flow conditions
  13. Statistical analysis
  14. Results
  15. Thrombin generation via rFVIIa enhances platelet deposition to collagen under flow conditions at normal and reduced platelet count
  16. Recombinant FVIIa increases maximal thrombus height at normal and reduced platelet count
  17. Time course of platelet deposition to collagen at reduced platelet count in absence or presence of thrombin generation
  18. rFVIIa-mediated thrombin generation enhances calcium signaling and exposure of procoagulant phospholipids after adhesion to collagen at normal and reduced platelet count
  19. rFVIIa binds to collagen-adhered platelets in normal blood and in blood from severely thrombocytopenic patients
  20. Thrombin generation via rFVIIa increases platelet adhesion and platelet spreading to fibrinogen under flow conditions at normal and reduced platelet counts
  21. Discussion
  22. Acknowledgements
  23. References

Intracellular calcium levels of single platelets during perfusion were determined as described previously [37]. In short, washed platelets were loaded with Fluo-3 acetoxymethylester (7 µm, 40 min incubation at room temperature). After centrifugation with ACD (1/10 v/v), platelets were resuspended in HEPES–Tyrode, pH 6.5. Subsequently, Fluo-3 platelets were mixed with non-labeled platelets (which were resuspended in HAS) and red cells to obtain reconstituted blood with a hematocrit of 40% and a platelet count of either 200 000 or 25 000 µL−1. Ten percent of the platelets in this reconstituted blood were labeled with Fluo-3. Perfusions were performed in the presence or absence of the thrombin-generating system for 5 min at a shear rate of 1600 s−1, and fluorescent images were recorded in real time using a Visitech digital imaging system (Sunderland, UK) equipped with a intensified charge-coupled device (CCD) camera. Changes in Fluo-3 fluorescence from individual platelets were converted to [Ca2+]i as described [38].

Determination of phosphatidylserine-exposing platelets after perfusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Proteins
  6. Collagen- and fibrinogen-coated surfaces
  7. Blood collection
  8. Perfusion studies
  9. Determination of maximal thrombus height
  10. Intracellular calcium measurements
  11. Determination of phosphatidylserine-exposing platelets after perfusion
  12. Binding of FITC-labeled rFVIIa to adhered platelets under flow conditions
  13. Statistical analysis
  14. Results
  15. Thrombin generation via rFVIIa enhances platelet deposition to collagen under flow conditions at normal and reduced platelet count
  16. Recombinant FVIIa increases maximal thrombus height at normal and reduced platelet count
  17. Time course of platelet deposition to collagen at reduced platelet count in absence or presence of thrombin generation
  18. rFVIIa-mediated thrombin generation enhances calcium signaling and exposure of procoagulant phospholipids after adhesion to collagen at normal and reduced platelet count
  19. rFVIIa binds to collagen-adhered platelets in normal blood and in blood from severely thrombocytopenic patients
  20. Thrombin generation via rFVIIa increases platelet adhesion and platelet spreading to fibrinogen under flow conditions at normal and reduced platelet counts
  21. Discussion
  22. Acknowledgements
  23. References

The procoagulant state of platelets adhered after 5 min of perfusion at 1600 s−1 was determined by perfusing FITC-labeled annexin A5 (0.1 µg mL−1) over collagen-adhered platelets for 1 min right after perfusion. After perfusing HEPES buffer for another 2 min, annexin A5 fluorescence and phase contrast images were recorded. Surface coverage and annexin A5 binding surface coverage were determined using Quanticell software (Visitech). To provide a measure of the proportion of deposited platelets expressing procoagulant surface, the ratio of annexin A5 positive surface to the total surface coverage measured by phase contrast was calculated as described [37].

Binding of FITC-labeled rFVIIa to adhered platelets under flow conditions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Proteins
  6. Collagen- and fibrinogen-coated surfaces
  7. Blood collection
  8. Perfusion studies
  9. Determination of maximal thrombus height
  10. Intracellular calcium measurements
  11. Determination of phosphatidylserine-exposing platelets after perfusion
  12. Binding of FITC-labeled rFVIIa to adhered platelets under flow conditions
  13. Statistical analysis
  14. Results
  15. Thrombin generation via rFVIIa enhances platelet deposition to collagen under flow conditions at normal and reduced platelet count
  16. Recombinant FVIIa increases maximal thrombus height at normal and reduced platelet count
  17. Time course of platelet deposition to collagen at reduced platelet count in absence or presence of thrombin generation
  18. rFVIIa-mediated thrombin generation enhances calcium signaling and exposure of procoagulant phospholipids after adhesion to collagen at normal and reduced platelet count
  19. rFVIIa binds to collagen-adhered platelets in normal blood and in blood from severely thrombocytopenic patients
  20. Thrombin generation via rFVIIa increases platelet adhesion and platelet spreading to fibrinogen under flow conditions at normal and reduced platelet counts
  21. Discussion
  22. Acknowledgements
  23. References

Direct binding of FVIIa to collagen-adhered platelets was investigated using rFVIIa, which was fluorescently labeled by means of a FITC-labeled active site inhibitor. FITC-labeled rFVIIa (2.4 µg mL−1) was added to whole blood anticoagulated with citrate and PPACK (Phe-Pro-Arg chloromethyl ketone, 40 µm), which was recalcified with 20 mm calcium chloride just before perfusion. The blood was perfused for 5 min over a collagen-coated coverslip, and immediately thereafter HEPES buffer was perfused for another 3 min. Subsequently, platelet-bound rFVIIa was visualized using two-photon laser scanning microscopy using a Bio-Rad 2100 multiphoton system (Hemel Hempstead, UK). Exitation was by a Spectra-Physics Tsunami Ti:Sapphire laser, tuned and mode-locked at 800 nm, producing pulses of ∼ 100 fs width (repitition rate 83 MHz). Scans were at 67% of maximal laser power at 166 lines per second. Fluorescence was detected at 508–523 nm.

Thrombin generation via rFVIIa enhances platelet deposition to collagen under flow conditions at normal and reduced platelet count

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Proteins
  6. Collagen- and fibrinogen-coated surfaces
  7. Blood collection
  8. Perfusion studies
  9. Determination of maximal thrombus height
  10. Intracellular calcium measurements
  11. Determination of phosphatidylserine-exposing platelets after perfusion
  12. Binding of FITC-labeled rFVIIa to adhered platelets under flow conditions
  13. Statistical analysis
  14. Results
  15. Thrombin generation via rFVIIa enhances platelet deposition to collagen under flow conditions at normal and reduced platelet count
  16. Recombinant FVIIa increases maximal thrombus height at normal and reduced platelet count
  17. Time course of platelet deposition to collagen at reduced platelet count in absence or presence of thrombin generation
  18. rFVIIa-mediated thrombin generation enhances calcium signaling and exposure of procoagulant phospholipids after adhesion to collagen at normal and reduced platelet count
  19. rFVIIa binds to collagen-adhered platelets in normal blood and in blood from severely thrombocytopenic patients
  20. Thrombin generation via rFVIIa increases platelet adhesion and platelet spreading to fibrinogen under flow conditions at normal and reduced platelet counts
  21. Discussion
  22. Acknowledgements
  23. References

The effect of in situ generation of thrombin on platelet deposition at normal platelet counts and at platelet counts representing thrombocytopenia was investigated by perfusing reconstituted blood consisting of red cells, and platelets in an albumin solution over collagen type III at a shear rate of 1600 s−1 for 5 min, in the presence or absence of a thrombin generating system. This thrombin generating system consists of coagulation factors FVIIa, FX and prothrombin, and was shown previously to result in thrombin generation on already adhered platelets [22]. As shown in Fig. 1A, platelet deposition progressively increased with increasing platelet count. On addition of the thrombin generating system, a statistically significant increased platelet surface coverage was observed for all platelet counts tested (between 10 000 and 200 000 platelets µL−1). Morphologically, a decrease in platelet count was associated with a decrease in platelet aggregate formation. Addition of the thrombin generating system increased aggregate size at all platelet counts. Representative micrographs of platelet thrombi formed at 200 000 and 25 000 platelets µL−1 in the presence or absence of the thrombin generating system are shown in Fig. 1B. When rFVIIa was omitted from the thrombin generating system, or if hirudin was added to the thrombin generating system, no increase in surface coverage or aggregate size was observed, indicating that the observed effects were indeed due to rFVIIa-mediated thrombin generation.

image

Figure 1. Thrombin generation via recombinant factor VIIa (rFVIIa) enhances platelet deposition to collagen under flow conditions at normal platelet count and under conditions representing thrombocytopenia. (A) Reconstituted blood containing different platelet counts was perfused over a collagen-coated surface for 5 min at a shear rate of 1600 s−1 in absence (closed symbols) or presence (open symbols) of coagulation factors rFVIIa (1.2 µg mL−1), factor (F)X (10 µg mL−1), and prothrombin (20 ng mL−1) in the presence of calcium chloride (3 mm). After perfusion, surface coverage was determined; the surface coverage values were normalized to the samples containing 200 000 platelets µL−1 in absence of clotting factors, which was set at 100%. The results shown represent five independent experiments, performed in duplicate. *P < 0.01; #P < 0.02; ^P = 0.03. Error bars indicate standard error of mean (SEM). (B) Morphological appearance of thrombi generated on a collagen surface with reconstituted blood at normal (200 000 µL−1) and reduced (25 000 µL−1) platelet count in absence or presence of the thrombin generating system (indicated as rVIIa/X/II). Shown are representative pictures of the experiments as described in A. Original magnification × 400.

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Recombinant FVIIa increases maximal thrombus height at normal and reduced platelet count

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Proteins
  6. Collagen- and fibrinogen-coated surfaces
  7. Blood collection
  8. Perfusion studies
  9. Determination of maximal thrombus height
  10. Intracellular calcium measurements
  11. Determination of phosphatidylserine-exposing platelets after perfusion
  12. Binding of FITC-labeled rFVIIa to adhered platelets under flow conditions
  13. Statistical analysis
  14. Results
  15. Thrombin generation via rFVIIa enhances platelet deposition to collagen under flow conditions at normal and reduced platelet count
  16. Recombinant FVIIa increases maximal thrombus height at normal and reduced platelet count
  17. Time course of platelet deposition to collagen at reduced platelet count in absence or presence of thrombin generation
  18. rFVIIa-mediated thrombin generation enhances calcium signaling and exposure of procoagulant phospholipids after adhesion to collagen at normal and reduced platelet count
  19. rFVIIa binds to collagen-adhered platelets in normal blood and in blood from severely thrombocytopenic patients
  20. Thrombin generation via rFVIIa increases platelet adhesion and platelet spreading to fibrinogen under flow conditions at normal and reduced platelet counts
  21. Discussion
  22. Acknowledgements
  23. References

To explore further the effects of rFVIIa-mediated thrombin generation on the morphology of a thrombus generated under flow conditions onto collagen type III, experiments were performed to determine the maximal thrombus height at different platelet numbers in the presence or absence of the thrombin generating system. Reconstituted blood with different platelet counts was perfused over glass coverslips coated with collagen type III in the presence or absence of coagulation factors FVIIa, FX and prothrombin for 5 min at a shear rate of 1600 s−1. After fixation, the thrombi were permeabilized, and the actin cytoskeleton was stained using TRITC-labeled phalloidin. Maximal thrombus height was determined by confocal laser scanning microscopy. As shown in Fig. 2, the platelet count had little effect on maximal thrombus height, although under this condition the amount of thrombi decreased progressively with decreasing platelet count (see Fig. 1). On addition of the thrombin generating system, maximal thrombus height was increased for all platelet counts examined (between 200 000 and 10,000 µL−1), but the difference did not reach statistical significance at 25 000 (P = 0.055) and 10 000 (P = 0.07) platelets µL−1.

image

Figure 2. Thrombin generation via recombinant factor VIIa (rFVIIa) results in higher thrombi formed on collagen under flow at normal and reduced platelet count. Reconstituted blood with different platelet counts was perfused over collagen at a shear rate of 1600 s−1 for 5 min in absence (open symbols) and presence (closed symbols) of a thrombin generating system as described in Fig. 1. Maximal thrombus height was determined using confocal scanning laser microscopy after permeabilization and staining with TRITC-labeled phalloidin. Results shown represent five independent experiments. *P < 0.01. Error bars indicate standard error of mean (SEM).

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Time course of platelet deposition to collagen at reduced platelet count in absence or presence of thrombin generation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Proteins
  6. Collagen- and fibrinogen-coated surfaces
  7. Blood collection
  8. Perfusion studies
  9. Determination of maximal thrombus height
  10. Intracellular calcium measurements
  11. Determination of phosphatidylserine-exposing platelets after perfusion
  12. Binding of FITC-labeled rFVIIa to adhered platelets under flow conditions
  13. Statistical analysis
  14. Results
  15. Thrombin generation via rFVIIa enhances platelet deposition to collagen under flow conditions at normal and reduced platelet count
  16. Recombinant FVIIa increases maximal thrombus height at normal and reduced platelet count
  17. Time course of platelet deposition to collagen at reduced platelet count in absence or presence of thrombin generation
  18. rFVIIa-mediated thrombin generation enhances calcium signaling and exposure of procoagulant phospholipids after adhesion to collagen at normal and reduced platelet count
  19. rFVIIa binds to collagen-adhered platelets in normal blood and in blood from severely thrombocytopenic patients
  20. Thrombin generation via rFVIIa increases platelet adhesion and platelet spreading to fibrinogen under flow conditions at normal and reduced platelet counts
  21. Discussion
  22. Acknowledgements
  23. References

To investigate the progression of platelet deposition to collagen in time in thrombocytopenic conditions, reconstituted blood containing 100 000 platelets µL−1 was perfused over a collagen-coated surface in the absence or presence of the thrombin generating system for 1.5, 3, 5, or 7.5 min. Platelet adhesion progressively increased over time, in both the absence and presence of the thrombin generating system. Enhancement of platelet deposition by addition of coagulation factors was observed only after 5 or 7.5 min of perfusion, but not at earlier time points (Fig. 3).

image

Figure 3. Time-course of platelet adhesion to collagen at reduced platelet count in absence and presence of thrombin generation. Reconstituted blood containing 100 000 platelets µL−1 was perfused over collagen at a shear rate of 1600 s−1 in absence (open symbols) or presence (closed symbols) of coagulation factors FVIIa, factor X, and prothrombin for 1.5, 3, 5, or 7.5 min. After perfusion, surface coverage was determined; shown are normalized values, the surface coverage of the samples perfused for 5 min in absence of clotting factors was set at 100%. The results shown represent three independent experiments, performed in duplicate. *P < 0.01. Error bars indicate standard error of mean (SEM).

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rFVIIa-mediated thrombin generation enhances calcium signaling and exposure of procoagulant phospholipids after adhesion to collagen at normal and reduced platelet count

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Proteins
  6. Collagen- and fibrinogen-coated surfaces
  7. Blood collection
  8. Perfusion studies
  9. Determination of maximal thrombus height
  10. Intracellular calcium measurements
  11. Determination of phosphatidylserine-exposing platelets after perfusion
  12. Binding of FITC-labeled rFVIIa to adhered platelets under flow conditions
  13. Statistical analysis
  14. Results
  15. Thrombin generation via rFVIIa enhances platelet deposition to collagen under flow conditions at normal and reduced platelet count
  16. Recombinant FVIIa increases maximal thrombus height at normal and reduced platelet count
  17. Time course of platelet deposition to collagen at reduced platelet count in absence or presence of thrombin generation
  18. rFVIIa-mediated thrombin generation enhances calcium signaling and exposure of procoagulant phospholipids after adhesion to collagen at normal and reduced platelet count
  19. rFVIIa binds to collagen-adhered platelets in normal blood and in blood from severely thrombocytopenic patients
  20. Thrombin generation via rFVIIa increases platelet adhesion and platelet spreading to fibrinogen under flow conditions at normal and reduced platelet counts
  21. Discussion
  22. Acknowledgements
  23. References

The effects of rFVIIa-mediated thrombin generation on the activation of platelets adhered to collagen were studied by real-time analysis of intracellular calcium responses of platelets adhered to collagen. In the absence of thrombin generation, a significant and sustained increase in intracellular calcium was observed after the platelets had firmly adhered. Increases in intracellular calcium were observed at physiological (200 000 µL−1, Fig. 4A) and thrombocytopenic (25 000 µL−1, Fig. 4C) platelet counts, although the calcium signal was lower at the reduced platelet count. On addition of the thrombin generating system, a substantial and sustained increase in intracellular calcium signal compared with the signal in the absence of thrombin generation was observed at both normal (Fig. 4B) and reduced (Fig. 4D) platelet count.

image

Figure 4. Intracellular calcium responses of collagen-adhered platelets in absence or presence of thrombin generation at normal and reduced platelet count. Reconstituted blood containing 200 000 (A,B) or 25 000 (C,D) platelets µL−1 was perfused over collagen at a shear rate of 1600 s−1 in absence (A,C) or presence (B,D) of coagulation factors FVIIa, factor X, and prothrombin. Part of the platelets were loaded with Fluo-3. Fluo-3 fluorescence was recorded during perfusion and the signal was converted to [Ca2+]i. Shown are averaged tracings of at least 20 different platelets from a single experiment, which is representative of three independent experiments.

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Subsequently, it was investigated whether the increased calcium response resulted in an increased exposure of procoagulant phospholipids. After 5 min of perfusion, binding of FITC-labeled annexin A5 to the adhered platelets was studied and compared with the total amount of platelets deposited onto the surface. Approximately 1% of the surface covered with platelets also stained positive for annexin A5 at both normal and reduced platelet count (Fig. 5), indicating that only a small fraction of the adhered platelets expressed substantial amounts of negatively charged phospholipids. On addition of the thrombin generating system, the amount of adhered platelets expressing negatively charged phospholipids was significantly enhanced at both normal and reduced platelet counts.

image

Figure 5. Procoagulant state of collagen-adhered platelets in absence or presence of thrombin generation at normal and reduced platelet count. Reconstituted blood containing 200 000 or 25 000 platelets µL−1 was perfused over collagen at a shear rate of 1600 s−1 in absence (control) or presence (VIIa/X/II) of coagulation factors FVIIa, factor X, and prothrombin. After 5 min perfusion, the coverslips were perfused with FITC-labeled annexin A5. Platelet-bound annexin A5 was visualized by fluorescence microscopy. Shown is the percentage of total surface coverage (as measured by phase contrast microscopy) also positive for annexin A5. *P < 0.05. Error bars represent standard error of mean (SEM), n = 3.

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rFVIIa binds to collagen-adhered platelets in normal blood and in blood from severely thrombocytopenic patients

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Proteins
  6. Collagen- and fibrinogen-coated surfaces
  7. Blood collection
  8. Perfusion studies
  9. Determination of maximal thrombus height
  10. Intracellular calcium measurements
  11. Determination of phosphatidylserine-exposing platelets after perfusion
  12. Binding of FITC-labeled rFVIIa to adhered platelets under flow conditions
  13. Statistical analysis
  14. Results
  15. Thrombin generation via rFVIIa enhances platelet deposition to collagen under flow conditions at normal and reduced platelet count
  16. Recombinant FVIIa increases maximal thrombus height at normal and reduced platelet count
  17. Time course of platelet deposition to collagen at reduced platelet count in absence or presence of thrombin generation
  18. rFVIIa-mediated thrombin generation enhances calcium signaling and exposure of procoagulant phospholipids after adhesion to collagen at normal and reduced platelet count
  19. rFVIIa binds to collagen-adhered platelets in normal blood and in blood from severely thrombocytopenic patients
  20. Thrombin generation via rFVIIa increases platelet adhesion and platelet spreading to fibrinogen under flow conditions at normal and reduced platelet counts
  21. Discussion
  22. Acknowledgements
  23. References

To show that rFVIIa is capable of binding to collagen-adhered platelets, after which it can initiate thrombin generation, experiments were performed in which FITC-labeled rFVIIa was added to whole blood from healthy volunteers and whole blood from severely thrombocytopenic patients. Whole blood anticoagulated with PPACK and citrate was recalcified, and after addition of FITC-labeled rFVIIa, the blood was perfused over a collagen-coated coverslip. After 5 min of perfusion, HEPES buffer was perfused over the coverslip for 3 min. Subsequently, bound FITC-labeled rFVIIa was visualized by sensitive two-photon fluorescence microscopy. As shown in Fig. 6A, after perfusion of blood from healthy volunteers, a substantial number of platelet aggregates showed FITC staining, indicating an interaction between FITC-labeled rFVIIa and the activated platelets. In the presence of EDTA, virtually no staining was observed (Fig. 6C). Also, when whole blood from five different patients with severe thrombocytopenia was used, FITC-labeled rFVIIa binding to the small platelet aggregates present on the collagen surface was demonstrated (a representative picture from a patient with a platelet count of 47 000 µL−1 is shown in Fig. 6B). The fluorescent threads, which are visible in all pictures, represent collagen fibers, which exhibited two-photon autofluorescence under the conditions used (data not shown).

image

Figure 6. Binding of recombinant factor VIIa (rFVIIa) to collagen-adhered platelets using whole blood from healthy volunteers (A) or severely thrombocytopenic patients (B). Citrated whole blood was recalcified in the presence of PPACK, and FITC-labeled rFVIIa was added. This blood was perfused over a collagen-coated surface for 5 min, and afterwards postperfused with HEPES buffer for 3 min. Subsequently, platelet-bound FITC–rFVIIa was visualized using fluorescence microscopy. As a control, whole blood was perfused in the presence of EDTA (50 mm, C). Shown are representative pictures from four experiments. Pictures represent 308 × 308 µm.

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Thrombin generation via rFVIIa increases platelet adhesion and platelet spreading to fibrinogen under flow conditions at normal and reduced platelet counts

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Proteins
  6. Collagen- and fibrinogen-coated surfaces
  7. Blood collection
  8. Perfusion studies
  9. Determination of maximal thrombus height
  10. Intracellular calcium measurements
  11. Determination of phosphatidylserine-exposing platelets after perfusion
  12. Binding of FITC-labeled rFVIIa to adhered platelets under flow conditions
  13. Statistical analysis
  14. Results
  15. Thrombin generation via rFVIIa enhances platelet deposition to collagen under flow conditions at normal and reduced platelet count
  16. Recombinant FVIIa increases maximal thrombus height at normal and reduced platelet count
  17. Time course of platelet deposition to collagen at reduced platelet count in absence or presence of thrombin generation
  18. rFVIIa-mediated thrombin generation enhances calcium signaling and exposure of procoagulant phospholipids after adhesion to collagen at normal and reduced platelet count
  19. rFVIIa binds to collagen-adhered platelets in normal blood and in blood from severely thrombocytopenic patients
  20. Thrombin generation via rFVIIa increases platelet adhesion and platelet spreading to fibrinogen under flow conditions at normal and reduced platelet counts
  21. Discussion
  22. Acknowledgements
  23. References

Next, the effect of thrombin generation on platelet adhesion to fibrinogen at a venous shear rate under normal and thrombocytopenic conditions was determined. Reconstituted blood containing different amounts of platelets was perfused over fibrinogen-coated coverslips at a shear rate of 300 s−1 for 5 min. As shown in Fig. 7A, platelet adhesion to fibrinogen progressively increased with increasing platelet count. Addition of the thrombin generating system resulted in a significant enhancement of surface coverage at all platelet counts tested. The thrombin generating system also substantially affected the morphology of fibrinogen-adhered platelets. In the absence of thrombin generation both spread platelets and contact platelets were observed at normal and reduced platelet counts. Addition of coagulation factors FVIIa, FX, and prothrombin substantially enhanced the number of spread platelets (Fig. 7B).

image

Figure 7. Thrombin generation via recombinant factor VIIa (rFVIIa) enhances platelet deposition to fibrinogen at normal and reduced platelet count. (A) Reconstituted blood with different platelet counts was perfused over a fibrinogen-coated surface at a shear rate of 300 s−1 for 5 min in absence (open symbols) and presence (closed symbols) of a thrombin generating system as described in Fig. 1. After perfusion, surface coverage was determined; the surface coverage values were normalized to the samples containing 200 000 platelets µL−1 in absence of clotting factors, which was set at 100%. The results shown represent five independent experiments, performed in duplicate. *P < 0.01; #P < 0.05. Error bars indicate standard error of mean (SEM). (B) Morphological appearance of thrombi generated on a fibrinogen surface with reconstituted blood at normal (200 000 µL−1) and reduced (25 000 µL−1) platelet count in absence or presence of the thrombin generating system (rFVIIa/X/II). Shown are representative pictures of the experiments as described in (A). Original magnification × 400.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Proteins
  6. Collagen- and fibrinogen-coated surfaces
  7. Blood collection
  8. Perfusion studies
  9. Determination of maximal thrombus height
  10. Intracellular calcium measurements
  11. Determination of phosphatidylserine-exposing platelets after perfusion
  12. Binding of FITC-labeled rFVIIa to adhered platelets under flow conditions
  13. Statistical analysis
  14. Results
  15. Thrombin generation via rFVIIa enhances platelet deposition to collagen under flow conditions at normal and reduced platelet count
  16. Recombinant FVIIa increases maximal thrombus height at normal and reduced platelet count
  17. Time course of platelet deposition to collagen at reduced platelet count in absence or presence of thrombin generation
  18. rFVIIa-mediated thrombin generation enhances calcium signaling and exposure of procoagulant phospholipids after adhesion to collagen at normal and reduced platelet count
  19. rFVIIa binds to collagen-adhered platelets in normal blood and in blood from severely thrombocytopenic patients
  20. Thrombin generation via rFVIIa increases platelet adhesion and platelet spreading to fibrinogen under flow conditions at normal and reduced platelet counts
  21. Discussion
  22. Acknowledgements
  23. References

The present study shows that thrombin generated via rFVIIa increases platelet deposition to collagen and fibrinogen under flow conditions. This increase in platelet deposition can be observed at normal and reduced platelet counts. These findings suggest that (part of) the mechanism of action of rFVIIa in thrombocytopenic patients can be attributed to enhancement of platelet deposition by increased (initial) thrombin generation, as has been suggested previously [26–28]. The elevated deposition of platelets is accompanied by enhanced activation of adhered platelets, as shown by increases in calcium fluxes, and potentiation of the procoagulant state of the deposited platelets. The enhanced generation of procoagulant phospholipid surface facilitates further enhancement of thrombin and fibrin generation. Enhancement of platelet adhesion and aggregation followed by increased fibrin generation induced by rFVIIa might compensate for the hemostatic defect which is present in thrombocytopenic patients, and this mechanism might explain the hemostatic effect of rFVIIa in these patients.

Analogous to previous experiments using platelets from patients with GT or normal platelets treated with an αIIbβ3-inhibiting peptide [22], the following sequence of events is postulated, which result increased hemostatic activity by addition of coagulation factors FVIIa, FX, and prothrombin. Initially, some platelets adhere to the surface, become activated, and expose procoagulant phospholipids. Subsequently, rFVIIa binds to these phosphatidylserine-exposing platelets, on which it activates FX independently of TF. The resulting FXa combines with factor V released from platelet α-granules, and the resulting prothrombinase complex converts prothrombin to thrombin. This in situ generated thrombin is a potent activator of platelets rolling on the surface or in flow. Thrombin activation of these platelets leads to enhanced platelet deposition and aggregation (on collagen) or spreading (on fibrinogen). Furthermore, this in situ generated thrombin contributes to platelet activation, resulting in higher calcium fluxes, and increased phosphatidylserine exposure in the adhered platelets.

Although the TF dependency of thrombin generation was not specifically addressed in this study, we have reason to believe the thrombin generation in our model is TF-independent. First of all, no TF is present on the surfaces used in this study (collagen and fibrinogen). Furthermore, enhancement of platelet adhesion to collagen is seen only after prolonged perfusion (5 or 7.5 min), indicating that the thrombin is generated on already activated platelets, and not already in suspension in which only unactivated platelets are present, which is in line with our previous observations with the same model of reconstituted blood, in which we investigated adhesion of platelets lacking functional αIIbβ3. In these studies, inhibitory antibodies against TF did not affect the enhancement of platelet deposition by rFVIIa-mediated thrombin generation [22]. In the same study, we also showed that rFVIIa could bind under flow conditions to normal platelets which adhered to a collagen surface. These findings were confirmed in the present study, and it was shown that rFVIIa is also able to bind collagen-adhered platelets under conditions of severe thrombocytopenia. Furthermore, in this study rFVIIa binding was investigated using a direct labeling approach, in contrast to indirect labeling via a mono- or polyclonal antibody which was used in previous studies [21,22]. Also, in a model in which we studied suspension aggregation of platelets lacking αIIbβ3 by rFVIIa-mediated fibrin formation, thrombin and fibrin generation via rFVIIa was independent of TF [30], which is in line with the results obtained in the cell-based model proposed by Hoffman, Monroe, and Roberts [39].

The exact mechanisms by which thrombin enhances platelet adhesion under flow conditions are incompletely known, but may include (a combination of) platelet activation by proteolysis of the protease activated receptors 1 and 4 (reviewed in [40]), proteolysis of glycoprotein (GP) V which makes the platelet more reactive to adhesive surfaces [41–43], and direct signaling through GPIb [41,44]. Furthermore, thrombin binding to GPIbα may directly result in enhancement of platelet–platelet interactions. The thrombin–GPIb crystal structure showed that two thrombin molecules bind to GPIb, one via exosite I and the other via exosite II. In this way, thrombin can bridge two GPIb molecules on different platelets, thereby contributing to aggregate formation [45,46]. It has to be noted, however, that a different crystal structure of thrombin and GPIb led to the conclusion that thrombin can cross-link two GPIb molecules on the same platelet, thus refuting a possible role of thrombin as a ligand supporting aggregation [47].

Enhancement of platelet adhesion and aggregation may not only (in part) explain the hemostatic efficacy of rFVIIa in patients with thrombocytopenia, but may be a general mechanism explaining part of the therapeutic effect of rFVIIa in hemophilia and the various novel indications. For all indications, enhancement of platelet adhesion and aggregation results in enhancement of primary hemostasis even at low platelet counts. Furthermore, enhancement of platelet adhesion and aggregation results in enhancement of procoagulant surface, thereby facilitating further thrombin and fibrin generation, leading to enhancement of secondary hemostasis.

Enhancement of secondary hemostasis (i.e. fibrin formation) under flow conditions by rFVIIa in different indications has been shown in a whole blood model [29,48,49]. However, no or little effect on platelet deposition was seen in these experiments. Two explanations may account for this apparent discrepancy in results. First, in this whole blood model, low-molecular-weight heparin (LMWH) was used as an anticoagulant. In LMWH anticoagulated blood endogenous FVII can be activated by factor XIIa formed by contact activation, and this phenomenon may reduce the effects of exogenous FVIIa on platelet deposition. Furthermore, it may be possible that the effects of rFVIIa on platelets are not observed in this model as a result of competition of platelets and fibrin for the surface. As fibrin formation is predominantly responsible for hemostasis at lower shear rates, it might be possible that in this model, the effects of rFVIIa on platelet do become apparent at higher shear rates. This would suggest that enhancement of platelet adhesion and aggregation predominantly contributes to the hemostatic effect of rFVIIa at higher shear rates.

In conclusion, our results suggest that administration of rFVIIa to thrombocytopenic patients and to patients with a normal platelet count enhances platelet deposition and platelet aggregation at the site of vascular injury. Furthermore, the deposited platelets are more strongly activated and show increased exposure of procoagulant surface, leading to a further enhancement of thrombin and fibrin generation. These phenomena might explain the hemostatic efficacy of rFVIIa in patients with thrombocytopenia, but could also be in part responsible for the beneficial effects of rFVIIa observed in patients with other hemostatic disorders.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Proteins
  6. Collagen- and fibrinogen-coated surfaces
  7. Blood collection
  8. Perfusion studies
  9. Determination of maximal thrombus height
  10. Intracellular calcium measurements
  11. Determination of phosphatidylserine-exposing platelets after perfusion
  12. Binding of FITC-labeled rFVIIa to adhered platelets under flow conditions
  13. Statistical analysis
  14. Results
  15. Thrombin generation via rFVIIa enhances platelet deposition to collagen under flow conditions at normal and reduced platelet count
  16. Recombinant FVIIa increases maximal thrombus height at normal and reduced platelet count
  17. Time course of platelet deposition to collagen at reduced platelet count in absence or presence of thrombin generation
  18. rFVIIa-mediated thrombin generation enhances calcium signaling and exposure of procoagulant phospholipids after adhesion to collagen at normal and reduced platelet count
  19. rFVIIa binds to collagen-adhered platelets in normal blood and in blood from severely thrombocytopenic patients
  20. Thrombin generation via rFVIIa increases platelet adhesion and platelet spreading to fibrinogen under flow conditions at normal and reduced platelet counts
  21. Discussion
  22. Acknowledgements
  23. References

The authors thank R. Røjkjær for providing rFVIIa. We acknowledge investment grant 902-16-276 from the Netherlands Organization for Scientific Research. Also, S. Moschatsis is gratefully acknowledged for her assistance in the initial stages of this study. This study was sponsored in part by an unrestricted educational grant of Novo Nordisk.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Proteins
  6. Collagen- and fibrinogen-coated surfaces
  7. Blood collection
  8. Perfusion studies
  9. Determination of maximal thrombus height
  10. Intracellular calcium measurements
  11. Determination of phosphatidylserine-exposing platelets after perfusion
  12. Binding of FITC-labeled rFVIIa to adhered platelets under flow conditions
  13. Statistical analysis
  14. Results
  15. Thrombin generation via rFVIIa enhances platelet deposition to collagen under flow conditions at normal and reduced platelet count
  16. Recombinant FVIIa increases maximal thrombus height at normal and reduced platelet count
  17. Time course of platelet deposition to collagen at reduced platelet count in absence or presence of thrombin generation
  18. rFVIIa-mediated thrombin generation enhances calcium signaling and exposure of procoagulant phospholipids after adhesion to collagen at normal and reduced platelet count
  19. rFVIIa binds to collagen-adhered platelets in normal blood and in blood from severely thrombocytopenic patients
  20. Thrombin generation via rFVIIa increases platelet adhesion and platelet spreading to fibrinogen under flow conditions at normal and reduced platelet counts
  21. Discussion
  22. Acknowledgements
  23. References