Reorganization of contractile elements in the platelet during clot retraction

doi:10.1016/S0022-5320(84)80027-1

Journal of Ultrastructure Research

Volume 89, Issue 1, October 1984, Pages 98-109

https://doi.org/10.1016/S0022-5320(84)80027-1 Get rights and content

Electron microscopic studies have been carried out on human platelets in the clot retraction. In the early stage of clot formation, platelets send out filopodia, in which thin filaments run longitudinally. The thin filaments are often observed to attach to the cell membrane where fibrin strands bind from the extracellular surface. In the later stage of clot formation, thick filaments become observable, mainly in the cell body of the platelets. These thick filaments are arranged to form an ordered array, and thin filaments run parallel to them. The thin filaments often attach to the end of the thick filaments. However, thin filaments are not seen between the arrays of thick filaments. Similar structures are also observed in the cytoskeleton of the contracted platelet. These filaments closely resemble the purified myosin aggregates formed under low ionic strength. Thus, during clot retraction, both actin and myosin in platelets are reorganized into thin and thick filaments, respectively.

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On the other hand, all taxane compounds, as well as ASA, showed a relatively negligible effect on ADP- or collagen-induced platelet aggregation as compared with AA-, U46619- or epinephrine-induced aggregation (data not shown). Platelets contain cytoskeletal networks of actin filaments [28,29], marginal microtubules [30], myosin molecules [31,32] and actin-binding proteins [33,34]. The contribution of the cytoskeleton in platelet aggregation has been reported.
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Myosin: A noncovalent stabilizer of fibrin in the process of clot dissolution
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Some observations support the possibility for association of fibrin with the platelet cytoskeleton.17 Around the fibrin fibers platelets send out filopodia, in which thick myosin filaments are arranged.18 In 2 hours after the initiation of thrombus formation morphologic signs of platelet necrosis are detected.
Myosin modulates the fibrinolytic process as a cofactor of the tissue plasminogen activator and as a substrate of plasmin. We report now that myosin is present in arterial thrombi and it forms reversible noncovalent complexes with fibrinogen and fibrin with equilibrium dissociation constants in the micromolar range (1.70 and 0.94 μM, respectively). Competition studies using a peptide inhibitor of fibrin polymerization (glycl-prolyl-arginyl-proline [GPRP]) indicate that myosin interacts with domains common in fibrinogen and fibrin and this interaction is independent of the GPRP-binding polymerization site in the fibrinogen molecule. An association rate constant of 1.81 × 10² M^–¹ · s^–¹ and a dissociation rate constant of 3.07 × 10^–⁴ s^–¹ are determined for the fibrinogen-myosin interaction. Surface plasmon resonance studies indicate that fibrin serves as a matrix core for myosin aggregation. The fibrin clots equilibrated with myosin are stabilized against dissolution initiated by plasminogen and tissue-type plasminogen activator (tPA) or urokinase (at fibrin monomer-myosin molar ratio as high as 30) and by plasmin under static and flow conditions (at fibrin monomer-myosin molar ratio lower than 15). Myosin exerts similar effects on the tPA-induced dissolution of blood plasma clots. Covalent modification involving factor XIIIa does not contribute to this stabilizing effect; myosin is not covalently attached to the clot by the time of complete cross-linking of fibrin. Thus, our in vitro data suggest that myosin detected in arterial thrombi binds to the polymerized fibrin, in the bound form its tPA-cofactor properties are masked, and the myosinfibrin clot is relatively resistant to plasmin.
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Activation of platelets leads to cytoskeletal assembly that is responsible for platelet motility and internal contraction. We have evaluated the involvement of the cytoskeleton in platelet activation by two strong agonists, collagen and thrombin. Activation was assessed by measuring changes in cytoskeletal assembly, externalization of activation-dependent markers and expression of procoagulant activity, and tyrosine phosphorylation of proteins, in both the absence and the presence of cytochalasin B. Activation of platelets with collagen and thrombin induced morphological changes and increased the expression of CD62P, CD63, glycoprotein IV, and binding of annexin V to platelets. Moreover, both activating agents induced actin polymerization, increased the association of other contractile proteins, and promoted tyrosine phosphorylation of multiple proteins, some of which were associated with the cytoskeleton. The presence of cytochalasin B blocked the previous events when collagen was used as the activating agent, although binding of annexin V still occurred. In contrast, platelet response to thrombin was not completely prevented by the presence of cytochalasin B. Thus, activation by collagen requires a functional cytoskeleton to trigger signaling through tyrosine phosphorylation and secretion. This is not the case for thrombin, which is capable of activating signaling mechanisms in the presence of strong inhibitors of cytoskeletal assembly. Moreover, the expression of a procoagulant surface in platelets still occurs even when platelet motility has been inhibited.
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α-Actinin and myosin were associated into reorganized actin cable networks and partly formed stress fiber-like structures in platelets during surface activation. Double-label immunofluorescence staining using antibodies against α-actinin and platelet myosin heavy chain (MHC) showed that α-actinin and myosin were colocalized in the cell center at the early stage of activation and dynamically redistributed with shape change. In the later stage, two proteins were colocalized around the granulomeres. α-Actinin was also seen beneath the surface membrane while myosin was not. Occasionally, both proteins were segregated, revealed granular staining in the cell body of flattened platelets and often aligned irregular alternate arrangement in the actin cables. Immunoelectron microscopy (immunogold) employing antibodies against MHC and myosin light chain (MLC) demonstrated that myosin, associated with actin cytoskeleton was precisely filamentous (328 nm in average length, 15 nm in width) and bipolar with a central bare zone, since MLCs were located at both ends. Myosin formed a cluster composed of several filaments with repeating alignment, suggesting each cluster corresponded to the granular staining pattern of immunofluorescence. These observations indicated that the organization of α-actinin and myosin in actin cables in activated platelets resembled that in stress fibers in various cultured cells.
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To study the localization of myosin in platelets, we utilized polyclonal antibody to heavy chain of platelet myosin. Both immunofluorescence and indirect immunogold staining techniques were employed. (1) In the unactivated platelets, myosin was distributed homogeneously throughout the cytosol. The cytosolic myosin was removed after platelets were treated with Triton X-100. The association of myosin with actin microfilaments in unactivated platelets was minimal. (2) In the surface-activated platelets, myosin was unextractable by Triton X-100. The myosin antibody heavily decorated the actin cable-networks which characterized the activated platelets. (3) In the Triton-unextractable cytoskeleton of both unactivated and activated platelets, we found fine fibrils (about 1-nm wide) that were often associated with immunogolds. These fibrils were similar to purified myosin molecules observed in rotary-shadowed metal replicas and ultrathin sections. These results indicate that cytosolic myosin becomes associated with actin cable-networks after the activation of platelets.
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Reorganization of contractile elements in the platelet during clot retraction

J. Ultrastruct. Res.

J. Biol Chem.

Biochem. Biophys. Res. Commun.

J. Biol Chem.

Exp. Cell Res.

Blood

Nature (London)

Trombos. Haemostasis

Nature (London)

Annu. Rev. Biochem.

Methods Achiev. Exp. Pathol.

Nature (London)

Thromb. Haemostasis