Arterial smooth muscle cell heparan sulfate proteoglycans accelerate thrombin inhibition by heparin cofactor II.
Heparin cofactor II (HCII) is a potent thrombin inhibitor in the presence of heparin and dermatan sulfate, glycosaminoglycans that accelerate the inhibition reaction. HCII is postulated to be an extravascular thrombin inhibitor that is stimulated physiologically by dermatan sulfate proteoglycans. To understand how thrombin activity may be downregulated within the artery wall, cultured monkey aorta smooth muscle cell (SMC) proteoglycans were tested for their ability to accelerate thrombin inhibition by HCII. Early confluent SMC monolayers increased thrombin-HCII inhibition rates 2-fold to 4-fold compared with reactions in cell-free control wells (7.3 +/- 0.5 versus 2.7 +/- 0.2 x 10(4) mol.L-1.min-1, with and without SMCs, respectively; n = 7 experiments). Extracellular matrix obtained by cell monolayer removal also accelerated the thrombin-HCII inhibition reaction 3-fold to 5-fold. Rate increases were abolished by Polybrene or protamine sulfate. Pretreatment of monolayers with heparitinase I (and of extracellular matrix with HNO2) to degrade heparan sulfate blocked the thrombin-HCII inhibition rate increase. In contrast, pretreatment with chondroitinase ABC in the presence of proteinase inhibitors had no effect. "Pericellular" (cell surface- and extracellular matrix-derived) SMC heparan sulfate proteoglycans (HSPGs) were purified and fractionated by charge on DEAE-Sephacel. At a concentration of 1 microgram/mL hexuronic acid, high-charge HSPG stimulated a 7-fold thrombin-HCII inhibition rate increase relative to reactions without proteoglycan, whereas low-charge HSPG induced a 2-fold rate increase. In comparison, an 18-fold rate increase was observed with 1 microgram/mL dermatan sulfate proteoglycan purified from SMC culture media. These results indicate that SMC HSPG could contribute significantly to thrombin inhibition by HCII in the artery wall.
Shirk
R A
RA
Department of Comparative Medicine, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, NC 27157-1040, USA.
Church
F C
FC
Wagner
W D
WD
D000818:Animals; D001013:Aorta, Thoracic / Q000378:metabolism; D002478:Cells, Cultured; D005109:Extracellular Matrix / Q000378:metabolism; D015844:Heparin Cofactor II / Q000378:metabolism* / Q000494:pharmacology; D006497:Heparan Sulfate / Q000378:metabolism* / Q000494:pharmacology; D008252:Macaca fascicularis; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism*
D015844:Heparin Cofactor II; D006497:Heparan Sulfate; D013917:Thrombin