Mechanobiology of Megakaryocyte-Driven Contraction of Plasma Clots
Authors: Kim Oleg , Litvinov Rustem I. , French Deborah L. , Brass Lawrence F. , Weisel John W. 
Published: December 2023

Background: Contractility of non-muscle cells plays a crucial role in various cellular processes including motility, morphogenesis, division, genome replication, intracellular transport, and secretion. Platelet-induced blood clot contraction is an essential process which plays a role in preventing bleeding and in thrombotic disorders. Megakaryocytes (MKs), which are the precursor cells of platelets, share many proteins and structures with platelets. However, little is known about the ability of MKs to contract and interact with extracellular matrix proteins such as fibrin. Previously, the non-muscle myosin II contractility was shown to be essential for MK migration within the bone marrow, to avoid premature proplatelet formation, and to allow branching of proplatelets. Recent studies have identified MKs in lung and brain tissues, as well as associated with thrombi in deceased COVID-19 patients. Notably, patients with severe COVID-19 exhibit elevated MK counts in their blood, indicating potential involvement in hemostasis and thrombosis. Nevertheless, the specific mechanisms by which MKs can contribute to clot contraction remain unknown.

Methods: Human induced pluripotent stem cells (iPSC) were differentiated into HPCs followed by expansion to megakaryocytes (iMKs) and analyzed by flowcytometry. Clot formation and contraction were initiated by adding thrombin and calcium to platelet-free-plasma with resuspended iMKs. A combination of real-time macroscale optical tracking, high-resolution confocal microscopy and biomechanical measurements were applied to elucidate the contractile cellular mechanisms and mechanotransduction pathways in iMKs, and ultimately comparing them with platelets.

Results: Flowcytometry analysis of unstimulated and thrombin-stimulated iMKs revealed that the major fraction of unstimulated cells expressed αIIbβ3 and GPIbα (~78%), but did not bind PAC-1, a monoclonal antibody specific for the activated conformation of αIIbβ3. Stimulation of iMKs with 1U/ml of thrombin resulted in PAC-1 binding (~68%), indicating activation of aIIbb3. The optical tracking of clot size change over time revealed that thrombin-stimulated iMKs were able to shrink macroscopic plasma clots with biphasic kinetics, similar to platelets. iMK-containing clots and platelet-reach-plasma clots equalized by the total cell surface area demonstrated comparable contraction rates and extent of contraction. The average maximal contractile force per individual iMK in the clot was found to be 145±98nN. In the presence of blebbistatin, the mean final extent and velocity of clot contraction of iMK-containing clots reduced in a dose-dependent manner, in comparison to untreated clots. Pretreatment of iMKs with 1μM latrunculin A, an inhibitor of actin polymerization, completely prevented contraction of plasma clots. In addition, contraction of plasma clots by activated iMKs was abrogated by 10μg/ml abciximab, that prevented binding of fibrin(ogen) to integrin a IIbb 3 receptors. The structural mechanisms of iMK contractility involved formation of filopodia and larger cytoplasmic protrusions undergoing extension-retraction cycles after being attached to surrounding fibrin fibers. Contraction of iMKs caused remodeling of the extracellular fibrin matrix by inducing spatial reorientation of fibrin fibers, accumulation of the fibrin mass on the iMK surface, and compaction of the entire fibrin network.

Conclusions: We have demonstrated a hitherto unknown ability of individual MKs to shrink fibrin clots and deciphered the cellular mechanisms of contractility of iPSC-derived MKs. The molecular mechanisms of MK-driven fibrin clot shrinkage were shown to be similar or identical to those of activated platelets, involving non-muscle myosin II activity, actin polymerization, and integrin αIIbβ3-fibrin interactions. Our findings provide a novel mechanistic insight into mechanobiology of MKs that may play a role in modulating the properties of hemostatic blood clots and thrombi. In addition, iMKs can be used as model cells with a potential of genetic modifications to study platelet structure and function, including their contractility.