Background: Blood clot contraction is mediated by platelets incorporated into the fibrin clot and driven by traction forces generated by the platelet cytoskeleton on the integrin αIIbβ3 that are transmitted to fibrin fibers bound to the αIIbβ3 extracellular domain. Deleting the gene for the cytosolic cysteine protease µ-calpain in mice impairs clot contraction, as does adding calpain inhibitors to human platelet-rich plasma.

Aims: To identify calpain-cleaved platelet proteins involved in clot contraction.

Methods: We used subtiligase-mediated biotin-labeling of nascent amino-termini and mass spectrometry.

Results: We identified 32 calpain cleaved proteins after TRAP stimulation. Many were cytoskeletal, most prominently talin and vinculin, whose complex constitutes a “mechanosensitive clutch” connecting integrins bound to the extracellular matrix to the actin cytoskeleton. Accordingly, we focused on talin and vinculin. Talin is composed of an N-terminal head domain attached to a C-terminal rod domain that is organized into a series of four- and five-helix bundles. These bundles contain 11 vinculin binding sites (VBS), each of which is an a-helix packed into a bundle interior. We detected 8 calpain-mediated cleavages in talin, 2 previously identified in unstructured regions and 6 in a-helical regions in proximity to a VBS. Because VBS are packed into the interior of a helical bundle, a structural rearrangement is required to initiate vinculin binding. There is substantial evidence in vitro that applying mechanical force across talin enables vinculin binding to the talin rod. However, we found that inhibiting platelet cytoskeletal contraction with blebbistatin or latunculin A had no effect on talin cleavage, indicating that talin cleavage by calpain in platelets does not require cytoskeleton-generated tensile force.

Conclusions: Based on these results, we propose that calpain-mediated cleavage of talin facilitates both vinculin binding to the talin rod and talin binding to αIIbβ3. These interactions then promote fibrin clot contraction caused by platelet cytoskeletal traction force.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/force-independent-cleavage-of-talin-by-calpain-promotes-platelet-mediated-fibrin-clot-contraction/