Cleavage of talin by calpain promotes platelet-mediated fibrin clot contraction.
Cleavage of talin by calpain promotes platelet-mediated fibrin clot contraction.
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钙蛋白酶对塔林的切割促进了血小板介导的纤维蛋白血块收缩。
DOI：
10.1182/bloodadvances.2021004582
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发表时间：
2021-12-14
期刊：
影响因子：
7.5
通讯作者：
Bennett, Joel S.
中科院分区：
文献类型：
--
作者：
Fong, Karen P.;Molnar, Kathleen S.;Agard, Nicholas;Litvinov, Rustem, I;Kim, Oleg, V;Wells, James A.;Weisel, John W.;DeGrado, William F.;Bennett, Joel S.
血小板中钙蛋白酶催化的塔林蛋白裂解促进纤维蛋白凝块收缩。钙蛋白酶在粘着斑蛋白结合位点附近切割塔林蛋白，可能启动粘着斑蛋白与塔林蛋白结合以促进凝块收缩。血凝块收缩由血小板细胞骨架产生的牵引力驱动，该牵引力通过整合素αIIbβ3传递至纤维蛋白纤维。在这里，我们表明，凝血收缩受损的血小板胞浆蛋白酶钙蛋白酶抑制剂。我们使用枯草杆菌酶介导的氨基末端标记和质谱法来鉴定参与凝块收缩的蛋白水解切割的血小板蛋白。在TRAP刺激后的32种钙蛋白酶切割蛋白中，有14种是细胞骨架蛋白，最突出的是talin和vinculin。talin和黏着斑蛋白的复合物构成机械敏感离合器，连接与细胞外基质结合的整合素和肌动蛋白细胞骨架。因此，我们集中研究了talin和vinculin。Talin由N-末端头部结构域和C-末端杆状结构域组成，它们被组织成一系列4-和5-螺旋束。该束含有11个黏着斑蛋白结合位点（VBS），每个位点都是一个α-螺旋，包装成束内部，需要结构重排才能启动黏着斑蛋白结合。我们在talin中检测到8个钙蛋白酶介导的切割，2个先前在非结构化区域中鉴定，6个在VBS附近的α-螺旋区域中。有证据表明，在体外施加机械力穿过talin使黏着斑蛋白结合到talin杆。然而，我们发现，抑制血小板细胞骨架收缩对talin裂解没有影响，这表明血小板中钙蛋白酶对talin裂解不需要细胞骨架产生的张力。因此，钙蛋白酶很可能通过局灶性粘连分解作用于凝块收缩的后期阶段。
Calpain-catalyzed talin cleavage in platelets promotes fibrin clot contraction. Calpain cleaves talin in proximity to vinculin binding sites, likely initiating vinculin binding to talin to promote clot contraction. Blood clot contraction is driven by traction forces generated by the platelet cytoskeleton that are transmitted to fibrin fibers via the integrin αIIbβ3. 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.
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