Activation of heparin cofactor II by fibroblasts and vascular smooth muscle cells.

E A Mcguire,D M Tollefsen

doi:10.1016/s0021-9258(19)75905-5

Abstract

Inhibition of thrombin by heparin cofactor II (HCII) is accelerated by dermatan sulfate, heparan sulfate, and heparin. Purified HCII or defibrinated plasma was incubated with washed confluent cell monolayers, 125I-thrombin was added, and the rate of formation of covalent 125I-thrombin-inhibitor complexes was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Fibroblasts and porcine aortic smooth muscle cells accelerated inhibition of thrombin by HCII 2.3-7.5-fold but had no effect on other thrombin inhibitors in plasma. Human umbilical vein endothelial cells and mouse macrophage-derived cells did not accelerate the thrombin-HCII reaction. IMR-90 normal human fetal lung fibroblasts treated with heparinase or heparitinase accelerated the thrombin-HCII reaction to the same degree as untreated cells. In contrast, treatment with chondroitinase ABC almost totally abolished the ability of these cells to activate HCII while chondroitinase AC had little or no effect, suggesting that dermatan sulfate was responsible for the activity observed. [35S]Sulfate-labeled proteoglycans were isolated from IMR-90 fibroblast monolayers and conditioned medium and fractionated into two peaks on Sepharose CL-2B. The lower Mr proteoglycans contained 74-76% dermatan sulfate and were 11-25 times more active with HCII than the higher Mr proteoglycans which contained 68-97% heparan sulfate. The activity of the lower Mr proteoglycans decreased 70-90% by degradation of the dermatan sulfate component with chondroitinase ABC. These results confirm that dermatan sulfate proteoglycans are primarily responsible for activation of HCII by IMR-90 fibroblasts. We suggest that HCII may inhibit thrombin when plasma is exposed to vascular smooth muscle cells or fibroblasts.

Highlights

Inhibition of thrombin by heparin cofactor I1 (HCII) activating protein C in the presence of thrombomodulin [2]
We find that HCII is activated by Thrombin, the serine protease derived from prothrombin fibroblasts and vascular smooth muscle cells but not by enduring blood coagulation, has a number of biologic activities. dothelial cells and macrophage-derived cells
NS, not significant ( p > 0.1). *, 0.05 >p > 0.02. **, p < 0.001, times and immediately assayed with 1251-thrombinand HCII or heparitinase, the results shown in Fig. 4A suggest that for complex formation (Fig. 4A).Preincubation with heparin- dermatan sulfate is responsible for the activation of HCII by ase or heparitinase had no effect on the amount of complex IMR-90 fibroblasts

Summary

RESULTS

Activation of HCII by Fibroblayts and Vascular Smooth Muscle Cells-We examined a variety of cells in monolayer culture for the ability to accelerate formationof the complex between'"I-thrombin andHCII. IMR-90 fibroblasts had noeffect on the rate of complex formation between '"'I-thrombin and ATIII (Fig. 2B). B, and d were not affected by the presence of cells, while 2.6 times morecomplexc was formedin the presence of cells In experiments with defibrinated plasma, smooth muscle cells only stimulated formation of the12511-thrombin-HCII complex (datanot shown). Smooth muscle cells did not accelerate complex formation betweenpurified ATIII and "'I-thrombin (data not shown). Defibrinated plasma and 1251-thrombinwere incubated with IMR-90 fibroblasts or empty wells as described in the legend to Fig. 3. 'Letters refer to bands labeled in Fig. 3. b p< 0.01 by Student's two-tailed t test in comparison to thecorresponding control ("-cells")

A Cell monolayer

B Cell extract

DISCUSSION