PB0357 - Megakaryocyte-Driven Blood Clot Contraction and its Structural Mechanisms

Background: Contraction of blood clots and thrombi is driven by platelets. A growing body of evidence suggests a direct involvement of megakaryocytes (MKs) in local blood clotting and thrombosis, but it is unknown whether MKs can drive contraction of blood clots and, if so, what are the mechanisms of MK-driven clot contraction.

Aims: To determine if MKs can induce contraction of clots and study dynamic structural alterations at the single-cell levels.

Methods: CD41(+)CD42b(+) MKs were generated from human induced pluripotent stem cells and suspended in citrated platelet-free human blood plasma at 4 million/mL. Clot formation and MK activation were induced by adding 2 U/ml thrombin and 3 mM CaCl2. Changes in clot size were followed for 60 min using an optical tracking system, and super-resolution confocal microscopy of MK-containing plasma clots was used to follow structural rearrangements of the fluorescently labeled cells and fibrin fibers.

Results: Thrombin-activated MKs caused contraction of plasma clots with the average final extent of contraction of ~50% and two kinetic phases that differed in the mean kinetic rates and durations. The contractility of MKs was sensitive to inhibitors of the myosin II ATPase activity, actin polymerization, and αIIbβ3-fibrin(ogen) binding, suggesting the same contractile machinery as in platelets. The biomechanical mechanism of MK-driven clot contraction involves formation of filopodia and larger cellular protrusions that attach to fibrin fibers and undergo extension-retraction cycles together with the cell body contraction. By pulling on the fibrin network, the contracting MKs actively remodel the clot, resulting in compaction of the entire MK-fibrin meshwork.

Conclusion(s): Our results provide evidence for the ability of MKs to contract fibrin clots and elucidate the structural mechanisms of MK-driven clot contraction, suggesting a novel mechanistic insight into the mechanobiology of MKs, which may play a direct role in modulating the permeability and obstructiveness of blood clots and thrombi.