Lai Katharine Crane, Flaumenhaft Robert
Center for Hemostasis and Thrombosis Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Cell Physiol. 2003 Feb;194(2):206-14. doi: 10.1002/jcp.10222.
In order to better understand the molecular mechanisms of platelet granule secretion, we evaluated the effect of activation-induced degranulation on three functional platelet SNARE proteins, SNAP-23, VAMP-3, and syntaxin 4. Initial studies showed that SNAP-23 is lost upon SFLLRN-induced platelet activation. Experiments with permeabilized platelets demonstrated that proteolysis of SNAP-23 was Ca(2+)-dependent. Ca(2+)-dependent proteolysis of SNAP-23 was inhibited by the cell-permeable calpain inhibitors, calpeptin and E-64d, as well as by the naturally occurring calpain inhibitor, calpastatin. In addition, purified calpain cleaved SNAP-23 in permeabilized platelets in a dose-dependent manner. In intact platelets, calpeptin prevented SFLLRN-induced degradation of SNAP-23. In contrast, calpeptin did not prevent SFLLRN-induced degradation of VAMP-3 and syntaxin 4 did not undergo substantial proteolysis following platelet activation. Calpain-induced cleavage of SNAP-23 was a late event occurring between 2.5 and 5 min following exposure of permeabilized platelets to Ca(2+). Experiments evaluating platelet alpha-granule secretion demonstrated that incubation of permeabilized platelets with 10 microM Ca(2+) prior to exposure to ATP inhibited ATP-dependent alpha-granule secretion from permeabilized platelets. SNAP-23 was cleaved under these conditions. Incubation of permeabilized platelets with either calpeptin or calpastatin prevented Ca(2+)-mediated degradation of SNAP-23 and reversed Ca(2+)-mediated inhibition of ATP-dependent alpha-granule secretion. Thus, activation of calpain prior to secretion results in loss of SNAP-23 and inhibits alpha-granule secretion. These studies suggest a mechanism whereby calpain activation serves to localize platelet secretion to areas of thrombus formation.
为了更好地理解血小板颗粒分泌的分子机制，我们评估了激活诱导的脱颗粒对三种功能性血小板SNARE蛋白SNAP - 23、VAMP - 3和 syntaxin 4的影响。初步研究表明，在SFLLRN诱导的血小板激活后，SNAP - 23会丢失。对透化血小板进行的实验表明，SNAP - 23的蛋白水解是Ca(2+)依赖性的。细胞可渗透的钙蛋白酶抑制剂钙肽素和E - 64d以及天然存在的钙蛋白酶抑制剂钙蛋白酶抑制蛋白均能抑制SNAP - 23的Ca(2+)依赖性蛋白水解。此外，纯化的钙蛋白酶以剂量依赖性方式在透化血小板中切割SNAP - 23。在完整血小板中，钙肽素可防止SFLLRN诱导的SNAP - 23降解。相比之下，钙肽素不能防止SFLLRN诱导的VAMP - 3降解，并且血小板激活后syntaxin 4未发生大量蛋白水解。钙蛋白酶诱导的SNAP - 23切割是一个晚期事件，发生在透化血小板暴露于Ca(2+)后2.5至5分钟之间。评估血小板α颗粒分泌的实验表明，在暴露于ATP之前，用10 microM Ca(2+)孵育透化血小板会抑制透化血小板中ATP依赖性α颗粒的分泌。在这些条件下，SNAP - 23被切割。用钙肽素或钙蛋白酶抑制蛋白孵育透化血小板可防止Ca(2+)介导的SNAP - 23降解，并逆转Ca(2+)介导的对ATP依赖性α颗粒分泌的抑制。因此，分泌前钙蛋白酶的激活导致SNAP - 23丢失并抑制α颗粒分泌。这些研究提示了一种机制，即钙蛋白酶激活有助于将血小板分泌定位到血栓形成区域。
