PURPOSE: Platelet-endothelial cell adhesion is an important pathologic response to vessel injury or inflammation. On binding to its endothelial or platelet G protein-linked seven-transmembrane domain receptor, protease-activated receptor-1 (PAR1), thrombin releases a 41-amino acid peptide (TR(1-41)). We examined the effect of TR(1-41) on platelet activation and on platelet-endothelial cell adhesion. METHODS: A monolayer of confluent human saphenous vein endothelial cells was incubated with washed human platelets. Platelets were stimulated with either TR(1-41), TR(21-41), scrambled TR(1-41), adenosine diphosphate (ADP)-epinephrine (EPI), thrombin, or thrombin receptor activating peptide (TRAP). Platelet activation was identified with flow cytometry. The magnitude of platelet-endothelial cell adhesion was determined with a laser scanning cytometer that scanned the monolayer of endothelial cells and identified fluorescently bound platelets. RESULTS: Maximal thrombin stimulation (0.1 U/mL) induced a threefold increase in platelets bound to endothelial cells compared with buffer alone. Stimulation with TR(1-41) (20 mmol/L) tripled the number of platelets bound to endothelial cells compared with thrombin. Scrambled sequence of TR(1-41) (20 mmol/L) and TR(21-41) (20 mmol/L), neither of which induces platelet activation, had minimal effect on platelet adhesion. Both TRAP (20 mmol/L) and ADP-EPI (20 mmol/L) induced less platelet-endothelial cell adhesion than did thrombin. TR(1-41)-induced platelet-endothelial cell adhesion was partially blocked by glycoprotein (GP)IIb-IIIa-specific monoclonal antibody, 10E5 (10 mg/mL). CONCLUSIONS: TR(1-41), the cleaved peptide of PAR1, is a more potent stimulant of platelet-endothelial cell adhesion than is thrombin, TRAP, or ADP-EPI, and this adhesion is at least in part mediated by the platelet GPIIb-IIIa receptor.

INTRODUCTION: An increased number of circulating platelet-monocyte aggregates (PMAs) is present in patients with all clinical classes of chronic venous insufficiency (CVI). The purpose of this study was to determine whether patients with CVI maintain elevated levels of PMAs following complete surgical correction of chronic venous insufficiency. METHODS: Patients with superficial venous insufficiency and a normal deep venous system documented by duplex scan were included in the study. Venous blood was drawn from a superficial vein in the leg and an antecubital vein prior to vein stripping and again six weeks postoperatively. Control subjects without evidence of venous disease had blood drawn from an antecubital vein. Whole blood flow cytometry was used to analyze the samples for the presence of platelet-monocyte aggregates following incubation with buffer or 0.5 microM adenosine diphosphate (ADP). RESULTS: Postoperative duplex scanning demonstrated elimination of venous reflux in the superficial venous system and normal deep vein physiology in all nine patients. Preoperatively, patients with CVI had significantly elevated levels of circulating PMAs in both arm and leg samples without stimulation by an agonist compared to controls (15.2+/-1.1 and 14.3+/-1.3 vs 7.4+/-0.3 for controls, pandlt;0.02 for each), and after stimulation by 0.5 microM ADP (33.7+/-4.7 and 34.3+/-5.2 vs 12.5+/-3.8 for controls, pandlt;0.04 for each). There was no significant change in the number of PMAs in either patient arm or leg blood samples six weeks following correction of venous reflux by removal of the diseased veins. CONCLUSIONS: Complete correction of chronic venous insufficiency did not diminish the elevated circulating levels of platelet-monocyte aggregates. We conclude that the presence of an increased number of PMAs identified in patients with CVI is not secondary to the presence of venous reflux, but may be involved with the primary etiology of chronic venous insufficiency. This finding also suggests that a stimulus other than venous hypertension may be important in triggering the leukocyte activation seen in patients with chronic venous disease.

BACKGROUND: Adherence of platelets to endothelial cells may be a significant event in the development of vascular thrombosis. Existing models, which examine platelet-endothelial cell interactions, compromise endothelial cell integrity or use radioactivity to identify platelets that adhere to endothelial cells. We report a novel method for in vitro detection of platelet-endothelial cell adhesion that allows endothelial cells to remain as an intact monolayer and for visualization of individual platelets. METHODS: Fluorescently labeled platelets were incubated with a confluent monolayer of endothelial cells. Laser scanning cytometry (LSC) identified platelets bound to endothelial cells based on their fluorescent signals. RESULTS: LSC detection of platelets reliably reproduced well-described findings of thrombin-induced platelet-endothelial cell adhesion. Results demonstrating reduced adhesion with a glycoprotein IIb-IIIa-specific blocking monoclonal antibody confirmed the specificity of the LSC detection of platelet-endothelial cell adhesion. CONCLUSIONS: LSC is a novel method for detecting platelet--endothelial cell adhesion. Its advantages over other methods are: (a) endothelial cells remain undisturbed and adherent throughout; (b) the ability to detect individual bound platelets and subpopulations; (c) the ability to store images and slides and then relocate, revisualize, and reanalyze individual cells or cell populations of interest; and (d) no radioactivity.

PURPOSE: Restenosis after angioplasty or bypass grafting to restore circulation to ischemic organs is still an unsolved problem. Thrombin generated in high concentrations at the sites of vascular injury plays a central role in thrombosis and hemostasis. alpha-Thrombin has also been implicated as a mitogen for smooth muscle cell (SMC) proliferation that contributes to arterial restenosis. Thrombomodulin has a high affinity of binding with thrombin and converts thrombin from a procoagulant to an anticoagulant. This study was designed to examine whether thrombomodulin could also moderate the thrombin-mediated SMC proliferative response. METHODS: Porcine carotid artery SMCs (passages 4-7) were plated onto 96-well plates and incubated for 3 days. After growth arrest in a defined serum-free medium for 2 to 3 days, SMCs were subjected to the reagents as follows: (1) human alpha-thrombin, (2) recombinant human soluble thrombomodulin containing a chondroitin sulfate moiety, (3) thrombin receptor agonist peptide (SFLLRNPNDKYEPF), and (4) alpha-thrombin or thrombin receptor agonist peptide combined with recombinant thrombomodulin (rTM). The viability and proliferation status of SMCs were quantified with MTT (thiazolyl blue) mitochondrial function and bromodeoxyuridine (BrdU)-DNA incorporation assays. RESULTS: Human alpha-thrombin increased SMC proliferation in a dose dependent manner by more than 25% and 30% with thrombin 1 U/mL to 3 U/mL compared with control groups on day 7 (PCONCLUSION: rTM containing all of the extracellular domains of thrombomodulin inhibits the effect of thrombin on SMC proliferation in vitro. Because thrombin is a mitogenic mediator of SMC in vascular injury, inhibition of its function in vivo could help to prevent SMC hyperplasia. The success of further studies in vivo may lead to use of rTM for decreasing or preventing arterial restenosis.