Quantification of platelet composition in experimental venous thrombosis by real-time polymerase chain reaction.
BACKGROUND
Platelets play a key role in thrombus formation. Determination of the platelet component in a thrombus provides pathophysiological insights to the thrombotic event and aids in selecting an appropriate therapeutic intervention. In this study a sensitive and reliable method to characterize the cellular components of experimental thrombi was developed using real-time polymerase chain reaction (PCR).


RESULTS
Vena cava thrombosis was induced by either oxidative injury to topical FeCl(2) (FeCl(2)-VT) or stenosis-limited blood flow and a hypotonic pressure stress (stasis-VT) in rats. High levels of platelets were identified in the thrombus containing vessels by real-time PCR analysis of target gene amplification using the 2(-DeltaDeltaCT) values by normalizing the data with gene expression in naive vessels and with a housekeeping gene, ribosomal protein L32. By this analysis, the levels of PF-4 (as a platelet marker) mRNA were significantly higher in FeCl(2)-VT (2(-DeltaDeltaCT)=7.8) than in stasis-VT (2(-DeltaDeltaCT)=4.2, p<0.05). Enhanced platelet enrichment in FeCl(2)-VT was also confirmed qualitatively by scanning electronic microscopic analysis. In addition, real-time PCR using a panel of genes representing vascular injury, inflammation and thrombosis showed marked induction (2(-DeltaDeltaCT)>5) in MCP-1, IL-1beta, iNOS and P-selectin mRNA expression in both models.


CONCLUSIONS
These data demonstrate the utility of real-time PCR to quantitate platelets and other cell components in vascular thrombosis, which may facilitate the characterization and thus therapeutic intervention of a particular thrombotic event in both preclinical animal models and clinical conditions.
Wang
Xinkang
X
Department of Thrombosis Biology, Bristol-Myers Squibb Company, Pennington, NJ 08534, USA. xinkang.wang@bms.com
Hsu
Mei-Yin
MY
Steinbacher
Thomas E
TE
Monticello
Thomas M
TM
Schumacher
William A
WA
D000818:Animals; D001792:Blood Platelets / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D018932:Chemokine CCL2 / Q000235:genetics; D002712:Chlorides; D004195:Disease Models, Animal; D005290:Ferric Compounds; D015870:Gene Expression / Q000187:drug effects; D053583:Interleukin-1beta / Q000235:genetics; D008297:Male; D008854:Microscopy, Electron; D052247:Nitric Oxide Synthase Type II / Q000235:genetics; D019007:P-Selectin / Q000235:genetics; D010978:Platelet Factor 4 / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012462:Saline Solution, Hypertonic; D013917:Thrombin / Q000032:analysis / Q000235:genetics; D013927:Thrombosis / Q000139:chemically induced / Q000235:genetics / Q000473:pathology; D014684:Venae Cavae / Q000293:injuries / Q000473:pathology; D020246:Venous Thrombosis / Q000139:chemically induced / Q000235:genetics* / Q000473:pathology
C491766:Ccl2 protein, rat; D018932:Chemokine CCL2; D002712:Chlorides; D005290:Ferric Compounds; D053583:Interleukin-1beta; D019007:P-Selectin; D012333:RNA, Messenger; D012462:Saline Solution, Hypertonic; D010978:Platelet Factor 4; D052247:Nitric Oxide Synthase Type II; D013917:Thrombin; C024555:ferric chloride