RESUMEN
ABSTRACT: Nonmuscle cell contractility is an essential feature underlying diverse cellular processes such as motility, morphogenesis, division and genome replication, intracellular transport, and secretion. Blood clot contraction is a well-studied process driven by contracting platelets. Megakaryocytes (MKs), which are the precursors to platelets, can be found in bone marrow and lungs. Although they express many of the same proteins and structures found in platelets, little is known about their ability to engage with extracellular proteins such as fibrin and contract. Here, we have measured the ability of MKs to compress plasma clots. Megakaryocytes derived from human induced pluripotent stem cells (iPSCs) were suspended in human platelet-free blood plasma and stimulated with thrombin. Using real-time macroscale optical tracking, confocal microscopy, and biomechanical measurements, we found that activated iPSC-derived MKs (iMKs) caused macroscopic volumetric clot shrinkage, as well as densification and stiffening of the fibrin network via fibrin-attached plasma membrane protrusions undergoing extension-retraction cycles that cause shortening and bending of fibrin fibers. Contraction induced by iMKs involved 2 kinetic phases with distinct rates and durations. It was suppressed by inhibitors of nonmuscle myosin IIA, actin polymerization, and integrin αIIbß3-fibrin interactions, indicating that the molecular mechanisms of iMK contractility were similar or identical to those in activated platelets. Our findings provide new insights into MK biomechanics and suggest that iMKs can be used as a model system to study platelet contractility. Physiologically, the ability of MKs to contract plasma clots may play a role in the mechanical remodeling of intravascular blood clots and thrombi.
Asunto(s)
Células Madre Pluripotentes Inducidas , Trombosis , Humanos , Megacariocitos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Plaquetas/metabolismo , Trombosis/metabolismo , Fibrina/metabolismo , PlasmaRESUMEN
Blood clot contraction is driven by traction forces generated by the platelet cytoskeleton that are transmitted to fibrin fibers via the integrin αIIbß3. Here we show that clot contraction is impaired by inhibitors of the platelet cytosolic protease calpain. We used subtiligase-mediated labeling of amino termini and mass spectrometry to identify proteolytically cleaved platelet proteins involved in clot contraction. Of 32 calpain-cleaved proteins after TRAP stimulation, 14 were cytoskeletal, most prominently talin and vinculin. A complex of talin and vinculin constitutes a mechanosensitive clutch connecting integrins bound to the extracellular matrix with the actin cytoskeleton. Accordingly, we focused on talin and vinculin. Talin is composed of an N-terminal head domain and a C-terminal rod domain organized into a series of 4- and 5-helix bundles. The bundles contain 11 vinculin binding sites (VBSs), each of which is an α-helix packed into a bundle interior and requiring structural rearrangement to initiate vinculin binding. We detected 8 calpain-mediated cleavages in talin, 2 previously identified in unstructured regions and 6 in α-helical regions in proximity to a VBS. There is evidence in vitro that applying mechanical force across talin enables vinculin binding to the talin rod. However, we found that inhibiting platelet cytoskeletal contraction had no effect on talin cleavage, indicating that talin cleavage by calpain in platelets does not require cytoskeleton-generated tensile force. Therefore, it is likely that calpain acts in the later stages of clot retraction through focal adhesion disassembly.
Asunto(s)
Talina , Trombosis , Sitios de Unión , Calpaína , Fibrina , Humanos , Talina/metabolismoRESUMEN
We have studied some biophysical properties of the fibrin network during the normal state of pregnancy and in patients with recurrent miscarriage (RM), in the first trimester of pregnancy. The fibrin polymerization process, followed by turbidity, showed that the rate of fibrin monomer assembly and the final turbidity was increased in the pregnant group (normal and with history of RM) compared to non-pregnant women (normal and RM), which is consistent with the increased fibrinogen concentration during pregnancy. No changes were observed in the Darcy constant (Ks) of RM clots, pregnant or not; however, in pregnant control subjects the Ks increased (p = 0.03). The fibrin lysis rate was increased in pregnant women compared to non-pregnant, being faster in women with RM. The rheological properties of the fibrin network in the non-pregnant group (control and RM patients) were similar; in the pregnant state, the fibrin network of the control group was 1.3 times stiffer compared to the control non-pregnant women, and almost unchanged in RM patients. In this study we have found changes in the clot structure that seem to be related to normal pregnancy and an increased rate of the fibrin lysis process in the RM patients, which may have clinical relevance.