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Activation of platelets in blood perfusing angioplasty-damaged coronary arteries. Flow cytometric detection
R E Scharf1, A Tomer, U M Marzec
1Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, Calif.
Insights
Platelets become activated in coronary arteries damaged by percutaneous transluminal angioplasty (PTCA). Flow cytometry effectively detects this platelet activation by measuring specific membrane glycoprotein epitopes.
Area of Science:
- Cardiovascular Biology
- Hematology
- Medical Diagnostics
Background:
- Percutaneous transluminal angioplasty (PTCA) can induce mechanical damage to coronary arteries.
- Platelet activation is a critical factor in thrombotic events following vascular injury.
Purpose of the Study:
- To investigate platelet activation in coronary arteries after PTCA using fluorescence-activated flow cytometry.
- To identify specific platelet activation markers detectable in blood flowing through damaged arteries.
Main Methods:
- Blood samples were collected from the coronary sinus before, during, and after PTCA in patients and controls.
- Fluorescence-activated flow cytometry utilized monoclonal antibodies (mAbs) targeting activation-dependent platelet epitopes (e.g., activated GPIIb-IIIa, P-selectin).
- Platelets and microparticles were identified using anti-glycoprotein (GP) Ib.
Main Results:
- Significant binding of activation-specific mAbs (PAC1, anti-LIBS1) to platelets was observed during and after PTCA.
- The proportion of platelets positive for PAC1 and anti-LIBS1 increased significantly post-PTCA compared to baseline.
- P-selectin expression was detected in a subset of patients; minimal activation markers were found in control groups.
Conclusions:
- Circulating platelets are activated when flowing through PTCA-damaged stenotic coronary arteries.
- Flow cytometry analysis of activation-specific membrane glycoprotein epitopes is a reliable method to demonstrate PTCA-induced platelet activation.
Abstract:
Fluorescence-activated flow cytometry has been used to investigate platelet activation in blood flowing through atherosclerotic coronary arteries after sustaining mechanical damage induced by percutaneous transluminal angioplasty (PTCA). For flow cytometry, platelets and platelet-derived microparticles were identified by biotinylated anti-glycoprotein (GP) Ib monoclonal antibody (mAb) and a fluorophore, phycoerythrin-streptavidin. Activated platelets were detected by using a panel of fluoresceinated mAbs specific for activation-dependent platelet epitopes, including 1) activated GPIIb-IIIa complex (PAC1); 2) fibrinogen bound to platelet GPIIb-IIIa (9F9); 3) ligand-induced binding sites on GPIIIa (anti-LIBS1); and 4) P-selectin, an alpha-granule membrane protein expressed on the platelet surface after secretion (S12). The binding of antibodies to platelets was determined in blood that was sampled continuously via heparin-coated catheters from the coronary sinus in 1) patients before, during, and for 30 minutes after PTCA and 2) control patients undergoing coronary angiography without PTCA. Platelets in coronary sinus blood showed significant binding of mAbs that specifically detect activation epitopes associated with the GPIIb-IIIa complex (PAC1, anti-LIBS1, and 9F9) during and for 30 minutes after angioplasty in four of the five patients. The relative proportion of platelets positive for PAC1 and anti-LIBS1 increased from baseline values of 2.0 +/- 0.3% (mean +/- SD) and 2.0 +/- 0.5% to 18 +/- 14% and 28 +/- 14%, respectively, during PTCA or 30 minutes after PTCA (p < 0.01 in both cases). Binding with 9F9 was less prominent. The expression of P-selectin was detected in one of the five patients. By contrast, activation-specific mAbs failed to bind detectably with platelets obtained from 1) the peripheral blood during coronary angiography in eight patients or 2) coronary sinus blood obtained via catheter throughout control catheterization procedures in three patients or before PTCA in five. We conclude that circulating platelets become activated while flowing through PTCA-damaged stenotic coronary arteries and that this process of platelet activation is readily demonstrated by measuring the expression of activation-specific membrane GP epitopes by flow cytometric analysis.