Cellular markers indicating activation of the hemostatic system : studies on platelets and leukocytes in peripheral human blood

Abstract

Platelet activation is supposed to initiate thrombus formation in many clinical settings such as coronary artery occlusions, stroke, diabetes mellitus complications, and in surgical interventions. Coagulation and thrombosis are also associated with inflammation in septicemia. The introduction of flow cytometry in the clinical laboratory allows studying of platelet and leukocyte activation in vivo associated with various clinical situations. A whole blood flow cytometry method with multiple fluorescent staining of platelets and leukocytes was adapted for simultaneous, quantitative measurement of platelet and leukocyte activation and estimation of leukocyte-platelet complexes.

A variant of the congenital bleeding disorder Bernard-Soulier syndrome, characterized biochemically by low expression of a structurally and functionally defective platelet glycoprotein receptor, GPIb(alpha), was identified by flow cytometry.

Quality control of platelet concentrates, in vitro, as platelet aggregation, adhesion and activation (degranulation) was determined when storage was prolonged for up to 9 days. The reduced expression of GP Ib(alpha) correlated positively with decreased von Willebrand factor binding capacity, indicating a progressive impairment of adhesiveness of platelet concentrates. Though there was no significant change in the expression of (alpha)IIb(beta)3, the fibrinogen binding capacity was significantly reduced, suggesting either a structural alteration of the receptor, or metabolic factors hampering the functional status of the platelets. Elevated P-selectin expression reflected a high degree of platelet activation during storage.

The time course for ligand binding (fibrinogen, von Willebrand factor) after exogenous stimulation was described for normal individuals. The fibrinogen binding was reversible after a maximum at 5 minutes after stimulation, possibly due to an internalisation of the receptor a few minutes after stimulation. Von Willebrand factor on the other hand increased, and remained at its maximum 30 minutes after stimulation. The expression of P-selectin was also stable after 40 minutes of development. Cardiopulmonary bypass causes no statistically significant platelet activation in form of changes of ligand binding or P-selectin expression. The large variation in these variables was partly due to the time differences on bypass.

The additive solution PAS-2 can be used as an alternative for storage of platelet concentrates because the storage of platelets in PAS-2 was comparable with autologous plasma, without significant decline in platelet function and viability.

A long-lasting effect on platelets and leukocytes after major surgery (hip arthroplasty) was found. Still 10 days after surgery thromboglobulin in plasma, monocyte-platelet and neutrophil-platelet complexes, prothrombin fragment 1+2, and CRP were significantly elevated compared to pre-surgery. These findings indicate a parallel activation and/or an interaction between the coagulation and immune systems. Monocyteplatelet and neutrophilplatelet complexes reflect hemostatic and inflammatory responses after surgery.

Signs of increased platelet activation (sP-selectin, PAC-I binding), leukocyte (CD11b CD64) activation as well as leukocyte-platelet interaction (CD11b+, CD64+ monocyte-platelet, CD11b+, CD64+ neutrophil-platelet complexes, monocyte-PAC-1+ platelet complexes) were demonstrated in patients with rheumatoid arthritis. These findings indicate a simultaneous activation of the immune and hemostatic systems. It might also to some extent explain the increased risk of thrombotic events in rheumatoid patients. TNF blocker therapy had no effect on platelet activation as PAC- I binding, suggesting that the patients, who are also indicated for risk of thrombotic events, need an appropriate therapeutic regime.

Taken together, our observations indicate the usefulness of flow cytometry for clinical analysis of platelet function and activation. It provides an accurate method not only for diagnosis of patients with platelet surface defects, but also for evaluation of platelet interaction with cells related to inflammatory disease, allowing determination of their pathophysiological significance/relevance in acute and chronic inflammatory diseases.