Background
Platelet-fibrin clot contraction is critical for wound closure and maintenance of vessel patency, yet a molecular understanding of the process has lagged because of a lack of flexible quantitative assay systems capable of assaying multiple samples simultaneously. Objectives: We devised a sensitive and inexpensive method to assess clot contraction kinetics under multiple conditions.
Conclusions
Our system is an adaptable platform for assessing the effects of multiple conditions and interventions on clot contraction kinetics in a regular laboratory setting, using readily available materials. The automated image processing software we developed will be made freely available for noncommercial uses. This assay system can be used to directly compare and define the effects of different treatments or genetic manipulations on platelet function and should provide a robust tool for future hemostasis/thrombosis research and therapeutic development.
Methods
Clot contraction was measured using time-lapse digital photography, automated image processing with customized software, and detailed kinetic analysis using available commercial programs.
Results
Our system was responsive to alterations in platelet counts and calcium, fibrinogen, and thrombin concentrations, and our analysis detected and defined three phases of platelet-fibrin clot formation: initiation, contraction, and stabilization. Lag time, average contraction velocity, contraction extent, and area under the curve were readily calculated from the data. Using pharmacological agents (blebbistatin and eptifibatide), we confirmed the importance of myosin IIA and the interactions of integrin αIIbβ3-fibrinogen/fibrin in clot contraction. As further proof of our system's utility, we showed how 2-deoxyglucose affects contraction, demonstrating the importance of platelet bioenergetics, specifically glycolysis. Conclusions: Our system is an adaptable platform for assessing the effects of multiple conditions and interventions on clot contraction kinetics in a regular laboratory setting, using readily available materials. The automated image processing software we developed will be made freely available for noncommercial uses. This assay system can be used to directly compare and define the effects of different treatments or genetic manipulations on platelet function and should provide a robust tool for future hemostasis/thrombosis research and therapeutic development.