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. 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.
|Journal||Research and Practice in Thrombosis and Haemostasis|
|State||Published - Jul 2022|
Bibliographical noteFunding Information:
The authors thank Dr. Jeremy P. Wood and the members of the Whiteheart Laboratory for their careful perusal of this manuscript and their patience. The authors also wish to thank Dr. John W. Weisel and his group for their help and specifically thank Drs. Valerie Tutwiler and Fazoil I. Ataullakhanov for demonstrating the HemaCore Thrombodynamics Analyzer System. The work was supported by grants from the NIH, NHLBI (HL56652, HL138179, and HL150818), and a Department of Veterans Affairs Merit Award to S.W.W. Q.J.W. was supported by pilot grants from the NIH, NIGMS COBRE (P30GM127211 and P20GM121327), NIDDK (P30 DK020579), NCRR/NACTS (UL1TR001998), and an AHA Grant‐in‐Aid.
© 2022 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH).
- clot contraction
- platelet function test
- platelet-rich plasma
- washed platelets
ASJC Scopus subject areas