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Keywords:

  • coagulation;
  • contact activation;
  • factor XIIa;
  • fibrinolysis;
  • kallikrein;
  • thromboelastography;
  • urokinase

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Summary. Background: The contact system (CS) proteins, factor XII and prekallikrein are thought to have roles in blood coagulation and fibrinolysis. Recent research has suggested that the CS proteins might be more important in fibrinolysis and cell function than in coagulation. Most studies on fibrinolysis have used plasma or euglobulin assays, ignoring the influence of cellular elements of blood on the fibrinolytic process. Objective and methods: In order to study both coagulation and fibrinolysis in whole blood (WB), we have developed a thromboelastography (TEG) assay to investigate both coagulation and fibrinolysis in the same blood sample. In this assay, named urokinase (UK) induced fibrinolysis in thromboelastography (UKIFTEG), TEG is performed on recalcified citrated WB in the presence of UK. Large variations in Ly60 (percentage lysis 60 min after clot formation) were obtained between different donors with the same UK concentration. The UKIFTEG assay was therefore performed using UK concentrations that gave Ly60 values in the approximate range of 20–40%. Results: The effect of CS activation was investigated in the presence or absence of celite (10 mg mL−1 blood). Celite shortened the clotting time (CT), and increased Ly60 values. Factor XIIa (FXIIa) and plasma kallikrein (KK) produced concentration dependent reductions in CT (significant at concentrations of 1303 and 2600 ng mL−1 blood, respectively) and increased Ly60 values (significant at concentrations of 652 and 1300 ng mL−1 blood, respectively). Conclusions: Our results show that CS activation and both FXIIa and KK produce reductions in clotting time and enhanced fibrinolysis in UKIFTEG.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Hemostasis (coagulation and fibrinolysis) involves the activities of blood cells, plasma proteins, phospholipids, venous endothelium and arterial intima. Pathophysiologic changes (acquired or inherited) in the balance of these activities can lead to bleeding or thrombosis.

The contact system (CS) proteins, factor XII (FXII), factor XI (FXI), prekallikrein (PK) and high molecular weight kininogen (HK) were identified as coagulation system proteins during the 1970s. Current consensus is that deficiencies in individual CS proteins (apart from FXI) do not lead to impaired coagulation in vivo [1]. The CS proteins have subsequently been shown to have roles in fibrinolysis, thrombin-induced platelet aggregation, cell adhesion, angiogenesis and blood pressure control [1,2,3]. Previous studies on the role of the CS proteins in fibrinolysis were performed using plasma or euglobulin fraction-based assays [4,5,6] and do not reflect the activities of FXII, PK and HK in whole blood.

The procedure of thromboelastography (TEG), originally described by Hartert in 1948 [7] not only allows for the measurement of global coagulation, but also yields data on the kinetics and dynamics of clot formation and clot lysis in whole blood. Modern versions of the TEG apparatus have subsequently been used in empirical screens of hemostatic function in liver transplantation and cardiopulmonary bypass [8,9]. Prior to these developments only the time until the onset of clotting could be measured and studies on coagulation and fibrinolysis in the same blood sample were difficult to perform.

In view of the important role of cellular elements of blood in hemostasis we have developed a TEG assay that enables the study of both coagulation and fibrinolysis in whole blood (WB). This assay, termed urokinase (UK) induced fibrinolysis in thromboelastography (UKIFTEG), involves the use of the plasminogen activator UK at critical concentrations to effect fibrinolysis in WB.

In the present communication the UKIFTEG model is described and its use to study the effects of CS activation and the addition of human factor XIIa (FXIIa) and KK on coagulation and fibrinolysis in WB reported.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Informed consent was obtained from the healthy subjects and the study approved by the Local Research Ethics Committee for East Kent.

Blood was obtained from healthy subjects with the minimum of stasis using a butterfly line and collected in siliconized Vacutainer® tubes (4.5 mL blood mixed with 0.5 mL 105 mm trisodium citrate; Becton Dickinson Ltd, Oxford, UK). The first 5 mL of blood was discarded. After gentle mixing, 1 mL volumes of citrated blood (WB) were distributed into several 1.5 mL non-wettable screw capped polypropylene tubes. Samples were left for 30 min at room temperature and mixed gently before analysis as recommended for TEG analysis [10].

For the celite activation studies, 1 mL volumes of WB were transferred to tubes that contained a final celite concentration of 10 mg mL−1 blood and the tubes capped and inverted five times before analysis.

The Thrombelastograph®, computer software, cups and pins, celite-containing tubes and calcium chloride (0.2 m) were purchased from Medicell Ltd, London, UK.

Full details of the 5000 series TEG® apparatus and TEG® analytical software can be obtained from the Haemoscope corporation web site at http://www.haemoscope.com. A brief summary of the TEG® apparatus and characteristics of the traces produced is as follows.

The major components of the TEG are cylindrical cups and pins (Fig. 1A). Cups warmed to 37 °C oscillate for 10 s through an angle of 4°45′ with pins freely suspended in the cups by torsion wires. Blood is added to the cups and torque is first transmitted as the clot forms and increases as the clot strengthens. As the clot lyses the torque decreases. A characteristic trace is obtained giving information on the rate of clot formation, clot strength and stability, and amount of fibrinolysis. The software automatically computes a coagulation index; a computer-derived classification of the traces. A schematic representation and interpretation of the TEG trace is shown in Fig. 1B. Four coagulation parameters are measured R, K, α and MA. R is the time from start of the sample run until the first detectable signs of clot formation. K is the time from the beginning of clot formation until a fixed level of clot firmness is obtained while α measures the rapidity of fibrin build-up and cross-linking (clot strength). MA or maximum amplitude is a measure of maximum strength of the clot and reflects the contribution of fibrin and platelets to the final strength of the clot. Fibrinolysis is determined by measuring the loss of clot strength with time after the MA is reached and is recorded as Ly30 and Ly60 (percentage lysis at 30 and 60 min after MA is reached). For a more detailed description and different interpretation of the TEG trace see references [9,11,12].

image

Figure 1. (A) Diagram of the thrombelastograph mechanism (B) Diagram and interpretation of a thromboelastograph (TEG®) tracing.

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Urokinase was purchased from Channel Diagnostics, Walmer, Kent, UK. It was calibrated against the First International Urokinase Standard obtained from the National Institute for Biological Standards and Control, Potters Bar, UK [activity: 4300 international units (IU) per ampoule], using an in ‘in-house’ chromogenic peptide substrate assay. Purified human FXIIa and KK were purchased from Enzyme Research Ltd, Swansea, UK. The enzymes were all dissolved in 0.9% sodium chloride.

UKIFTEG was performed as follows: The TEG® apparatus was set up according to the manufacturer's instructions and calibrated each day with the relevant calibrators. For the studies on contact activated and non-contact activated blood and following set up, the following procedure was used: to the cups (prewarmed to 37 °C) was added; either 5 μL 0.9% NaCl or UK at various concentrations and 20 μL of 0.2 m calcium chloride. Finally, 335 μL celite-activated WB or non-celite treated WB samples were added, the pins inserted into the cups and the tracing started. The TEG traces were run for up to 90 min.

For the studies with FXIIa and KK the above procedure was followed with the exception that 50 μL of FXIIa or KK (at various concentrations) were added to the cups before the calcium chloride followed by 300 μL WB (without celite). For the studies with FXIIa and KK without UK the above procedure was followed with the exception that the 5 μl UK was replaced with 5 μL 0.9% NaCl.

Fibrinolysis was expressed as Ly30 or Ly60. These equate to the calculated percentage of clot lysed 30 and 60 min after maximum clot strength has been reached. Activities of UK and concentrations of FXIIa and KK were calculated as IU mL−1 and ng mL−1 WB, respectively.

Statistical analyses

Statistical analysis was carried out using Statistica Mac (Statsoft Ltd, Uckfield, UK). Statistical significance was set at a P-value of <0.05 and calculated using t-test.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

The effect of varying UK activities on whole blood fibrinolysis

In an initial study, UK was added to citrated blood from a healthy subject to give final activities of between 0 and 86 IU mL−1. Some of the resulting TEG traces are shown in Fig. 2. Very little lysis was seen with UK up to 58 IU mL−1 blood. A sudden increase in percentage lysis 30 and 60 min after clot formation (Ly30 and Ly60) was observed between 64 and 71 IU mL−1 of UK. Subsequently TEG was performed on WB from 15 male and 15 female donors without and with three levels of UK (58, 66 and 71 IU mL−1). The results for Ly30 and Ly60 values are shown in Fig. 3. These results show that in the absence of UK very little fibrinolysis was found in normal recalcified citrated blood from the 30 subjects studied. In the presence of UK large variations in Ly30 and Ly60 values were found in different healthy subjects at the same UK concentrations. There was no significant difference in the results obtained for male and female donors.

image

Figure 2. Thromboelastograph tracing of recalcified citrated whole blood from a single donor in the absence and presence of urokinase (UK) at various concentrations. (A) No UK, (B) UK 58 IU mL−1 blood, (C) UK 64 IU mL−1 blood, (D) UK 71 IU mL−1 blood.

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image

Figure 3. Effect of various urokinase concentrations on fibrinolysis (Ly30 and Ly60%) in whole blood samples from 30 healthy subjects. Results expressed as mean ± SD%.

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The effect of celite activation on coagulation and fibrinolysis in UKIFTEG

The UKIFTEG assay was performed on blood samples from five male and five female healthy subjects. Each blood sample was first assayed to find a level of UK that gave a reasonable fibrinolytic activity and then tested with either celite activated or non-activated WB in the presence of the chosen UK concentration. The results are shown in Fig. 4. As expected celite activation produced a pronounced and highly significant (P = <0.0001) reduction in clotting time (CT, mean R ± SD non-activated and activated WB 12.68 ± 2.97 min and 6.22 ± 1.01 min, respectively). Celite activation also produced enhanced fibrinolysis (Ly60 mean ± SD% non-activated and activated WB, 11.18 ± 4.42% and 16.99 ± 6.32%, respectively P = 0.028). Typical UKIFTEG traces for non-activated and celite activated WB are shown in Fig. 5A and B. The traces were similar in blood samples from all healthy subjects.

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Figure 4. Effect of celite activation on coagulation and fibrinolysis in the urokinase induced fibrinolysis in thromboelastography model. NCFIB, Non-celite activation fibrinolysis (Ly60%); CFIB, celite activation fibrinolysis (Ly60%); NCCT, non-celite activation coagulation (clotting time; R min); CCT, celite activation coagulation (clotting time; R min). Results expressed as mean ± SD.

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image

Figure 5. Urokinase induced fibrinolysis in thromboelastography tracings of: (A) Non-activated and (B) Celite activated whole blood.

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The effect of FXIIa and KK on UKIFTEG

The UKIFTEG assay was performed on blood samples from six healthy subjects (three male and three female) in the absence or presence of various concentrations of FXIIa (0–20 850 ng mL−1 WB) and KK (0–20 800 ng mL−1 WB). The results for CT (R) and fibrinolysis (Ly60) are shown in Figs 6A–B and 7A–B. Both FXIIa and KK produced dose dependent reductions in CT (significant at concentrations of 1303 and 2600 ng mL−1 WB, respectively) and fibrinolysis (significant at concentrations of 652 and 1300 ng mL−1 WB), respectively.

image

Figure 6. Effect of a range of concentrations of factor XIIa (FXIIa) on (A) coagulation (clotting time; R min) and (B) fibrinolysis (Ly60%) in urokinase induced fibrinolysis in thromboelastography on blood from six healthy subjects. Results expressed as mean ± SD. Significance values were calculated for each FXIIa concentration by comparison with the 0 group.

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image

Figure 7. Effect of a range of concentrations of plasma kallikrein (KK) on (A) coagulation (clotting time; R min) and (B) fibrinolysis (Ly60%) in urokinase induced fibrinolysis in thromboelastography on blood from six healthy subjects. Results expressed as mean ± SD. Significance values were calculated for each KK concentration by comparison with the 0 group.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

One of the major impediments to investigating the effects of CS activated proteins on fibrinolysis in whole blood has been the lack of a suitable model for studying the activities. Various procedures have been described for detecting fibrinolysis in blood including the dilute blood clot lysis assay [13], the whole blood clot lysis assay [14] and the use of radiolabeled fibrinogen [15]. However very little data is available on studies with CS activation or activated CS proteins. We have therefore developed the modified TEG® model described in this article for this purpose.

When citrated WB is recalcified and subjected to TEG the computer controlled TEG® records the characteristic trace (Fig. 2, trace A). This trace can be broken down into several components reflecting various aspects of clot formation and subsequent lysis (Fig. 1B). As can be seen by trace A in Fig. 2 very little fibrinolysis occurs in non-CS activated blood from a healthy subject. We have therefore added the fibrinolytic enzyme UK to induce a degree of fibrinolysis and make the assay more sensitive to changes to the fibrinolytic process. We have named this model UKIFTEG. We chose UK as the fibrinolytic activator because the CS has fibrinolytic links with UK (1).

We observed that in the UKIFTEG model very little fibrinolysis is produced by UK at activities up to 58 IU mL−1 blood and that a sudden increase in fibrinolysis occurs at certain UK concentrations that differ from subject to subject. A typical TEG® trace for one subject is shown in Fig. 2. In this subject the sudden increase in lysis occurred between 64 and 71 IU mL−1 (Traces C and D). A similar observation of a sudden increase in fibrinolysis in a plasma-based tPA induced fibrinolysis model has recently been reported [12]. When blood samples from 30 donors (15 male and 15 female) were tested in the UKIFTEG assay using three UK concentrations large variations in Ly30 and Ly60 values were obtained (Fig. 3). These results probably reflect the variations in the combined effects of platelet number, total fibrin formed, degree of cross linkage within the clot, as well as the concentrations of plasminogen and fibrinolytic inhibitors (α2-plasmin inhibitor and PAI-1) present in each individual blood sample. For routine and research studies the UKIFTEG assay is run at UK concentrations that give Ly60 values in the approximate range 20–40%. Above UK concentrations of 58 IU mL−1 blood the model becomes very sensitive to small increases in fibrinolysis and as little as 8 IU mL−1 UK over the 58 IU mL−1 has a demonstrable fibrinolytic effect (Figs 2 and 3).

Activation of the CS was reported to stimulate fibrinolysis in the 1960s and 1970s and elegant studies by Kluft et al. [5] on plasma and euglobulin preparations suggested that CS proteins constitute approximately 50% to the total fibrinolytic activity in plasma. Both activated factor XII (FXIIa) and KK can affect fibrinolysis both directly by converting plasminogen to plasmin [16,17,18] and indirectly whereby KK converts pro-UK [single chain UK plasminogen activator (scu-PA)] to UK [two-chain UK plasminogen activator (tcu-PA)] [19]. In whole blood fibrinolysis is more complicated. Cellular effects on fibrinolysis include the priming of polymorphonuclear leukocytes (PMN) by KK for activation and release of PMN enzymes such as elastase and cathepsin G, which have fibrin(ogen)olytic activities [1]. In addition both u-PA and its receptor u-PAR are stored in PMN granules and PMN have been reported to be the central cell-type for spontaneous lysis for model thrombi [20].

PK and scu-PA are both associated with platelets [21,22,23] and it has been postulated that contact dependent fibrinolysis is mediated by platelets at the site of a forming thrombus [22].

In current routine monitoring studies TEG® assays are run following celite activation which reduces both the TEG® running time and result variability. As celite functions by activating the CS, we examined the effect of celite activation on coagulation and fibrinolysis using blood samples from five male and five female donors in the UKIFTEG model. A significant increase (P = 0.028) in fibrinolysis (Ly60 value) was obtained (Fig. 4). Celite is highly negatively charged and when added to blood not only activates the various CS proteins but also affects platelets and other blood cells. Interestingly the UKIFTEG trace in the presence of celite (Fig. 5) is similar to the traces obtained in blood samples from patients with stage 1 disseminated intravascular coagulation, showing reduced CT, platelet activation and increased clot strength followed by secondary fibrinolysis [24].

The advantage of the UKIFTEG model is that it can be used to investigate the effects of purified enzymes and other plasma components on coagulation and fibrinolysis in blood from individual subjects. To our knowledge such studies using purified preparations of FXIIa and KK have not been previously reported. Our results on blood samples from small populations of healthy subjects indicate that both enzymes reduce CT and increase fibrinolysis in a dose dependent manner in the model. Significant reductions in CT were produced by 1303 ng FXIIa and 2600 ng KK per mL WB and significant increases in fibrinolysis by 652 ng FXIIa and 1300 ng KK per mL WB (Figs 6A and 7A and 6B and 7B, respectively). As a portion of the added enzymes would be bound to and inhibited by C1-esterase inhibitor and other inhibitors (1) it is probable that the actual amounts of active enzymes available for coagulation and fibrinolysis are markedly less than the amounts added to the blood. It must be born in mind however that the blood samples used in our studies already contain FXII and PK so it is probable that activation of these intrinsic proenzymes by either FXIIa or KK will lead to production of some ‘in sample’ FXIIa and KK somewhat balancing the effect of plasma inhibitors.

Of relevance to our results are the observation of Nielsen et al, [25], that the TEG® coagulation profiles for a hereditary angioedema patient (C1-esterase inhibitor deficiency) with higher than normal plasma levels of FXII were similar to the ones we obtained with added FXIIa (low R-value, and high values for α, MA and clot strength).

Interestingly in TEG® using WB from three donors and in the absence of UK (results not shown), FXIIa and KK produced similar reductions in CT to those found in the presence of UK. The effects on fibrinolysis were however minimal even at FXIIa and KK concentrations above 5200 ng mL−1 WB. These results are not suprising in view of our observation that UK activities below 58 IU mL−1 WB also produced very little fibrinolysis (Fig. 2.) Our results confirm that both FXIIa and KK induce coagulation in WB in our in vitro model and that a synergism exists between the CS enzymes and UK with respect to fibrinolysis in whole blood. This relationship is probably mediated through platelets [21,22] and possibly PMN's.

FXII and PKK circulate at plasma concentrations of between 15–47 ug mL−1 and 35–50 ug mL−1, respectively (1). Levels of the active enzymes FXIIa and KK are very low in plasma samples from healthy donors [equivalent to conversions of FXII and PKK of <1% (M. J. Gallimore, D. W. Jones and H. P. Wendel unpublished results)]. The concentrations of active enzymes in the UKIFTEG model, which gave significant reductions in CT, were equivalent to conversions of blood levels of between 2.8–8.7% of FXII and 5.2–7.4% of PKK and for fibrinolysis of between 1.4–4.3% of FXII and 2.6–3.7% of PKK. It is probable that conversions of these magnitudes could occur under pathophysiologic conditions such as cardiopulmonary bypass and septic shock with FXIIa and KK contributing to the hypercoagulation and fibrinolysis often observed in these situations.

Subjects deficient in FXII, PK or HK do not generally have a bleeding tendency, therefore these proteins are not thought to have major roles in modern models of coagulation [26]. In fact following the death of the index case for FXII deficiency (Mr Hageman) from a thromboembolus [27] it has been suggested that inherited or acquired deficiencies of these proteins may be risk factors for thrombosis [1,28]. Although this is disputed for FXII deficiency [29], our results on the fibrinolytic activities of FXIIa and KK in our WB model, present the possibility that in some situations the CS proteins could have a role in thrombolysis.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References
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