ACTIVE SITE-INHIBITED RFVIIA BLOCKS PLATELET STRING FORMATION ON INTACT ENDOTHELIUM ACTIVATED WITH TNF-ALPHA AND SHIGA TOXIN-1

Abstract number: P-M-292

Grabowski¹ E.F., Nestoridi¹ E., Kushak² R., Ingelfinger³ J.R.

¹¹Pediatric Hematology Oncology ²²Pediatric Gastroenterology ³³Pediatric Nephrology, Harvard Medical School, Boston, United States

How-to-cite Grabowski EF, Nestoridi E, Kushak R, Ingelfinger JR. ACTIVE SITE-INHIBITED RFVIIA BLOCKS PLATELET STRING FORMATION ON INTACT ENDOTHELIUM ACTIVATED WITH TNF-ALPHA AND SHIGA TOXIN-1. J Thromb Haemost 2007; 5 Supplement 2: P-M-292

Abstract

Introduction: Human blood flowing over monolayers of endothelial cells (ECs) in vitro under controlled flow conditions is an exciting model of blood-EC interactions which also permits real-time imaging of single platelets (1 micron resolution) and platelet strings and aggregates. We have developed such a model to study platelet adhesion/aggregation on monolayers of human renal microvascular ECs grown on optically clear vinyl slides and activated with TNFalpha (20 ng/ml; hr 0-22) and Shiga toxin-1 (Stx; 10 pM; hr 18-22), in simulation of events in vivo in the childhood epidemic hemolytic uremic syndrome (HUS).

Methods: Normal donor blood, collected into 4 U/ml low MW heparin (dalteparin) and the Tab anti-GPIIb moAb and an ALEXA 555-conjugated second antibody, was drawn at shear rates of 270-650 sec^-1 through a parallel-plate flow chamber of which one wall was one of the above slides. Shear stresses approximated the 25 dynes/cm² estimated for glomerular arterioles. Imaging was via real-time epifluorescence digital videomicroscopy.

Results: With activated, but not control ECs, clusters of platelets adhered to the monolayers in strings tethered at the upstream end, and aggregates. In 5 paired experiments, monolayer preincubation x 30 min with 60 nM active site-inhibited rFVIIa (ASIS; courtesy of Dr. Ulla Hedner, Novo Nordisk) eliminated the strings and aggregates, reducing platelet depostion (% image pixels) from 16 4.2 to 1.8 0.54 (P<0.01), as well as local fibrin formation (immunostaining). Single platelet adhesion (to constitutively expressed and upregulated ICAM-1 and other integrins/selectins) was unaffected.

Conclusions: Platelet string and aggregate formation on activated endothelium is TF-driven, as shown by its inhibition by both ASIS and anti-TF moAb. Importantly, ASIS does not block single platelet adhesion. This and other anti-TF pathway agents may have promise as therapies for the prothrombotic and proinflammatory effects of epidemic HUS.

To cite this abstract use the following format:

Journal of Thrombosis and Haemostasis 2007; Volume 5, Supplement 2: abstract number

Session Details

Date:	01/08/2007
Time:	00:00-00:00
Session name:	XXIst ISTH Congress
Subject:
Location:	Oxford, UK
Presentation type:
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