Role of the membrane skeleton in preventing the shedding of procoagulant-rich microvesicles from the platelet plasma membrane.

The platelet plasma membrane is lined by a membrane skeleton that appears to contain short actin filaments cross-linked by actin-binding protein. Actin-binding protein is in turn associated with specific plasma membrane glycoproteins. The aim of this study was to determine whether the membrane skeleton regulates properties of the plasma membrane. Platelets were incubated with agents that disrupted the association of the membrane skeleton with membrane glycoproteins. The consequences of this change on plasma membrane properties were examined. The agents that were used were ionophore A23187 and dibucaine. Both agents activated calpain (the Ca2(+)-dependent protease), resulting in the hydrolysis of actin-binding protein and decreased association of actin with membrane glycoproteins. Disruption of actin-membrane interactions was accompanied by the shedding of procoagulant-rich microvesicles from the plasma membrane. The shedding of microvesicles correlated with the hydrolysis of actin-binding protein and the disruption of actin-membrane interactions. When the calpain-induced disruption of actin-membrane interactions was inhibited, the shedding of microvesicles was inhibited. These data are consistent with the hypothesis that association of the membrane skeleton with the plasma membrane maintains the integrity of the plasma membrane, preventing the shedding of procoagulant-rich microvesicles from the membrane of unstimulated platelets. They raise the possibility that the procoagulant-rich microvesicles that are released under a variety of physiological and pathological conditions may result from the dissociation of the platelet membrane skeleton from its membrane attachment sites.

Identification of a membrane skeleton in platelets.

Platelets have previously been shown to contain actin filaments that are linked, through actin-binding protein, to the glycoprotein (GP) Ib-IX complex, GP Ia, GP IIa, and an unidentified GP of Mr 250,000 on the plasma membrane. The objective of the present study was to use a morphological approach to examine the distribution of these membrane-bound filaments within platelets. Preliminary experiments showed that the Triton X-100 lysis buffers used previously to solubilize platelets completely disrupt the three-dimensional organization of the cytoskeletons. Conditions were established that minimized these postlysis changes. The cytoskeletons remained as platelet-shaped structures. These structures consisted of a network of long actin filaments and a more amorphous layer that outlined the periphery. When Ca2+ was present, the long actin filaments were lost but the amorphous layer at the periphery remained; conditions were established in which this amorphous layer retained the outline of the platelet from which it originated. Immunocytochemical experiments showed that the GP Ib-IX complex and actin-binding protein were associated with the amorphous layer. Analysis of the amorphous material on SDS-polyacrylamide gels showed that it contained actin, actin-binding protein, and all actin-bound GP Ib-IX. Although actin filaments could not be visualized in thin section, the actin presumably was in a filamentous form because it was solubilized by DNase I and bound phalloidin. These studies show that platelets contain a membrane skeleton and suggest that it is distinct from the network of cytoplasmic actin filaments. This membrane skeleton exists as a submembranous lining that, by analogy to the erythrocyte membrane skeleton, may stabilize the plasma membrane and contribute to determining its shape.

Evidence that agonist-induced activation of calpain causes the shedding of procoagulant-containing microvesicles from the membrane of aggregating platelets.

One of the responses of platelets to stimulation is activation of intracellular calpain (the Ca(2+)-dependent protease). Previously, we have shown that activation of calpain in platelets is involved in the generation of platelet procoagulant activity. Because procoagulant activity is present on the microvesicles that are shed from activated platelets, in this study we examined whether calpain is involved in the shedding of microvesicles. Platelets were incubated with the physiological agonists collagen or thrombin. The extent of activation of calpain correlated positively with the amount of procoagulant-containing microvesicles that formed, and the shedding of procoagulant-containing microvesicles was inhibited by calpeptin, MDL, and EST (E-64-d), three membrane-penetrating inhibitors of calpain. The protein composition of the microvesicles shed from aggregating platelets was similar to that of microvesicles shed by platelets in which the association of the membrane skeleton with the plasma membrane had been disrupted by incubation of platelets with dibucaine or ionophore A23187. Furthermore, like microvesicles shed from dibucaine- or ionophore A23187-treated platelets, those shed from the aggregating platelets possessed procoagulant activity. These results are consistent with the possibility that activation of calpain in aggregating platelets causes the shedding of procoagulant-containing microvesicles. We suggest that the shedding of microvesicles results from the calpain-induced hydrolysis of the platelet membrane skeleton.

Effects of ionic strength and state of assembly on kinetics of hydrogen exchange of calf thymus histones.

The kinetics of hydrogen exchange of calf thymus histone H2A-H2B dimers and (H3-H4)2 tetramers at pH 7 have been examined at low (0.16 M NaCl) and high (2 M NaCl) ionic strengths and after incorporation into (H2A-H2B-H3-H4)2 octamers. The similarity of the results for both species is noteworthy. Approximately 60% of the backbone amide protons are detectable in both low and high salt, and at least three kinetic phases can be distinguished. Increasing the ionic strength from 0.16 to 2 M accelerates exchange of some of the rapidly exchanging protons in both dimers and tetramers, while slowing exchange of others. Exchange of the more slowly exchanging protons is virtually unaffected. Incorporation of dimers into octamers accelerates exchange of approximately 40 protons to such an extent that they can no longer be detected. The effects of assembly upon the tetramer are qualitatively similar. These results indicate that both high ionic strengths and assembly destabilize some regions of the structure while stabilizing others. For both dimers and tetramers, the effects of ionic strength are dramatic, while those of assembly are more subtle. Higher resolution studies aimed at identifying the responsive protons would be of interest.