doi: 10.1161/01.STR.0000163112.92158.22
(Stroke. 2005;36:929-a.)
© 2005 American Heart Association, Inc.
Letters to the Editor |
In Vitro Models for Assessing Transcranial Ultrasound-Enhanced Thrombolysis
Department of Neurology, University of Heidelberg, Universitätsklinikum Mannheim, Mannheim, Germany
Hemostasis/Coagulation Division, Department of Medicine, University of Heidelberg, Universitätsklinikum Mannheim, Mannheim, Germany
To the Editor:
Contrary to the promising results of recent clinical studies, Pfaffenberger et al conclude from in vitro data that diagnostic ultrasound (US) is not suitable for transcranial enhancement of tissue plasminogen activator (tPA) thrombolysis.1 Their contention is based on surprising control data showing significant clot lysis without tPA and plasminogen and relatively modest increases in clot dissolution with tPA. Interestingly, their reported clot weight loss of 21.8% after 1 hour of incubation in saline alone is much higher than that which they recently reported in another journal using the same methods (2.8%±1.8%2 and 11.0%±3.8%3). Likewise, the effect of tPA alone (27.4%) also differs with earlier results of 19.9%±4.3%3 and 22.7%±9.0%.2 These discrepancies seem important, because a 19.9% effect of tPA alone would have been lower than combined US and recombinant tPA through the skull (26.2%), thus encouraging the authors to positively assess transcranial US thrombolysis.
Because clot dissolution is dependent on the amount of plasminogen present and on the amount of tPA available for activation of the plasminogen, little clot lysis should occur during the first 60 minutes of incubation in saline, with or without tPA. To study our hypothesis, we used experimental conditions similar to those of Pfaffenberger et al. 1.5 mL of citrated whole blood was mixed with 60 µL of 756 µmol/L CaCl2 and incubated for 1 hour at 37°C. Clots were weighed and transferred to polypropylene tubes containing 11 mL of saline, saline with a final concentration of 8.62 µg/mL recombinant tPA (Actilyse; Boehringer Ingelheim, Germany), or 86.2 µg/mL tPA. Clots were weighed again after 1 hour and 24 hours. D-dimer antigen was measured after 1 hour and 24 hours using reagents and methods from BioMérieux, Durham, NC.
As expected, we found no significant clot weight loss after 1 hour of incubation, even in the presence of the high concentration of tPA. Values for clot weight loss and D-dimer antigen after 1 and 24 hours are shown in the Table.
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Without added rtPA, only a very small proportion of plasminogen is activated by plasminogen activators present within the clot. At the higher concentration of rtPA, plasminogen activation occurs more rapidly, but the maximal plasmin activity is not greater than at the lower rtPA concentration. This is because a high concentration of tPA inactivates some of the plasmin generated, resulting in a lower degree of clot dissolution.
In vitro models are useful for studying different mechanisms of US-enhanced thrombolysis. The contribution of each mechanism will differ, depending on its importance on lysis rate in the specific model used. Pfaffenberger et al use an in vitro model that does not adequately support the study of either physiological tPA lysis or US enhancement of thrombolysis. Ultrasound is known to facilitate the entry of tPA into clots, resulting in more rapid generation of plasmin. If plasminogen is present in the surrounding medium, ultrasound also enhances the entry of plasminogen, resulting in high plasmin activity within the clot and more rapid clot dissolution. Without addition of plasminogen, in vitro tPA lysis of human clot in saline shows no significant activity after 1 hour of incubation. Therefore, clot weight losses of 20% to 30% cannot be explained by proteolytic degradation and do not constitute meaningful data for questioning the merits of recent clinical trials providing evidence for US enhancement of tPA thrombolysis in stroke patients.
References
1. Pfaffenberger S, Devcic-Kuhar B, Kollmann C, Kastl SP, Kaun C, Speidl WS, Weiss TW, Demyanets S, Ullrich R, Sochor H, Wober C, Zeitlhofer J, Huber K, Groschl M, Benes E, Maurer G, Wojta J, Gottsauner-Wolf M. Can a commercial diagnostic ultrasound device accelerate thrombolysis? An in vitro skull model. Stroke. 2005; 36: 124–128.
2. Pfaffenberger S, Devcic-Kuhar B, El Rabadi K, Groschl M, Speidl WS, Weiss TW, Huber K, Benes E, Maurer G, Wojta J, Gottsauner-Wolf M. 2MHz ultrasound enhances t-PA-mediated thrombolysis: comparison of continuous versus pulsed ultrasound and standing versus travelling acoustic waves. Thromb Haemost. 2003; 89: 583–589.[Medline] [Order article via Infotrieve]
3. Devcic-Kuhar B, Pfaffenberger S, Gherardini L, Mayer C, Groschl M, Kaun C, Benes E, Tschachler E, Huber K, Maurer G, Wojta J, Gottsauner-Wolf M. Ultrasound affects distribution of plasminogen and tissue-type plasminogen activator in whole blood clots in vitro. Thromb Haemost. 2004; 92: 980–985.[Medline] [Order article via Infotrieve]
Response:
Department of Internal Medicine II, University of Vienna, Waehringer Guertel, Austria
In answer to Dr Meairs’ letter concerning transcranial in vitro models for ultrasound-enhanced thrombolysis, we emphasize that the reason for varying control groups in 3 published studies1–3 by our group is a completely different sonication set-up reflecting the diverse study aims (schematic descriptions of the respective set-ups are shown as figures in 2 studies1,3). The sonication methods were therefore not the same, as stated, and resulted in different control levels.
The authors of the letter produced whole blood clots to evaluate clot lysis. As described in their letter, clots should be weighed after l hour and 24 hours. However, as shown in a Table, the crucial parameter of clot weight after l hour of treatment was not measured, but only estimated according to D-dimer levels. Because of the high standard deviation up to nearly 100%, this method seems to be unreliable. In addition, a more accurate time response curve would have been helpful to get a better understanding of the presented data for 2 reasons. First, clots after l hour revealed a weight loss of 0.46%, but after 24 hours the weight loss was 19.82%, which is about 43x higher. This is somehow curious. Second, why are high concentrations of recombinant tissue plasminogen activator more effective after l-hour treatment but less effective after 24-hour treatment when compared with low recombinant tissue plasminogen activator concentrations? The authors state that this actually interesting effect is caused by inactivation of some of the generated plasmin at high recombinant tissue plasminogen activator levels. However, they do not provide the reader with any reference to support this notion.
As stated by the authors, ultrasound is known to facilitate the entry of tissue plasminogen activator into clots, which was demonstrated by our group recently,4 resulting in more rapid generation of plasmin. Plasminogen is present within the whole blood clot, as demonstrated in the same study by immunostaining. We agree with the authors that long-term treatment of in vitro clots, for instance, over 24 hours, would benefit from plasminogen substitution, but more relevant short-term effects are also mediated by locally present plasminogen.
However, as demonstrated in several studies, lysis rates of older clots are significantly reduced even if plasminogen is added.5,6 This might be because of the fact that internal plasminogen is then already consumed. The authors further state that ultrasound enhances the entry of plasminogen into the clot when added to the surrounding medium. This of course would be an important point, but again there is no reference given confirming this information.
In conclusion, we think that our study does not constrain the importance and significance of a recently published multicenter trial.7 As discussed in the article, in vitro data cannot entirely explain in vivo outcomes for stated reasons. However, we believe that information gained from our data might be useful for further designs of basic studies or even in vivo trials in this field.
References
1. Pfaffenberger S, Devcic-Kuhar B, Kolhnann C, Kastl SP, Kaun C, Speidl WS, Weiss TW, Demyanets S, Ullrich R, Sochor H, Wober C, Zeitlhofer J, Huber K, Groschl M, Benes E, Maurer G, Wojta J, Gottsauner-Wolf M. Can a commercial diagnostic ultrasound device accelerate thrombolysis? An in vitro skull model. Stroke. 2005; 36: 124–128.
2. Pfaffenberger S, Devcic-Kuhar B, El-Rabadi K, Groschl M, Speidl WS, Weiss TW, Huber K, Benes E, Maurer G, Wojta J, Gottsauner-Wolf M. 2MHz ultrasound enhances t-PA-mediated thrombolysis: comparison of continuous versus pulsed ultrasound and standing versus travelling acoustic waves. Thromb Haemost. 2003; 89: 583–589.[Medline] [Order article via Infotrieve]
3. Devcic-Kuhar B, Pfaffenberger S, Groschl M, Kollmann C, Benes E, Gottsauner-Wolf M. In vitro thrombolysis enhanced by standing and travelling ultrasound wave fields. Ultrasound Med Biol. 2002; 28: 1181–1187.[CrossRef][Medline] [Order article via Infotrieve]
4. Devcic-Kuhar B, Pfaffenberger S, Gherardini L, Mayer C, Groschl M, Kaun C, Benes E, Tschachler E, Huber K, Maurer G, Wojta J, Gottsauner-Wolf M. Ultrasound affects distribution of plasminogen and tissue-type plasminogen activator in whole blood clots in vitro. Thromb Haemost. 2004; 92: 980–985.[Medline] [Order article via Infotrieve]
5. Luo H, Steffen W, Cercek B, Arunasalam S, Maurer G, Siegel RJ. Enhancement of thrombolysis by external ultrasound. Am Heart J. 1993; 125: 1564–1569.[CrossRef][Medline] [Order article via Infotrieve]
6. Olsson SB, Johansson B, Nilsson AM, Olsson C, Roijer A. Enhancement of thrombolysis by ultrasound. Ultrasound Med Biol. 1994; 20: 375–382.[CrossRef][Medline] [Order article via Infotrieve]
7. Alexandrov AV, Molina CA, Grotta JC, Garami Z, Ford SR, Alvarez-Sabin J, Montaner J, Saqqur M, Demchuk AM, Moye LA, Hill MD, Wojner AW; CLOTBUST Investigators. Ultrasound-enhanced systemic thrombolysis for acute ischemic stroke. N Engl J Med. 2004; 351: 2170–2178.
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