ADAMTS (a disintegrin and metalloproteinase domain, with thrombo spondin type-1 modules) is a family of 19 secreted Zn-metalloproteinases, which have multidomain structural components in common [1] . These include an N-terminal signal peptide, followed by a pro-domain, a metalloproteinase catalytic domain with a zinc binding motif, a disintegrin-like domain, a central thrombospondin type-1-like repeat (TSR), a cysteine rich domain (high sequence homology), a spacer region, and a variable number of C terminal TSR repeats. This family plays important roles in several pathophysiological conditions mainly including arthritis [2] , spermatogenesis [3] , angiogenesis [4] [5] , and thrombosis-related disease [6] . Noteworthily, most of these activities are related to proteolytic processing within their C-terminal regions [6] - [9] .

ADAMTS-18 has the similar domain organization as other family members. ADAMTS-18 has been shown to be epigenetically silenced in multiple carcinomas and has tumor suppressor activity [10] . Mutation of ADAMTS-18 is strongly associated with colorectal cancer [11] . The data from National Center for Biotechnology Information (NCBI) subject’s gene expression omnibus (GEO) also showed that ADAMTS-18 gene was differentially expressed in subjects with normal skeletal fracture versus subjects with nonunion skeletal fracture [12] . Therefore, it is also associated with bone mineral density (BMD) determination in the major human ethnic groups. Recently, some studies indicate that the ADAMTS-18 gene is also play a crucial role in early eye development [13] .

Platelet integrin αIIbβ3 (GPIIb/IIIa) is a heterodimeric receptor of the integrin family expressed at high density (50,000 - 80,000 copies/cell) on the platelet plasma membrane [14] . GPIIIa49-66 (CAPESIEFPVSEAREVLED) is a linear epitope of integrin subunit β3 (GPIIIa) in its extracellular domain. We previously reported that the unique feature of the antibodies (Abs) against GPIIIa49-66 was their ability to induce reactive oxygen species (ROS) through the activation of 12-lipoxygenase and nicotinamide adenine dinucleotide phosphate oxidase (NADPH), leading to complement-independent platelet fragmentation [15] [16] . Recently, we revealed that ADAMTS-18 was the physiologic ligand of platelet GPIIIa49-66 [17] . Thrombin generated from the endothelium of vessel injury is able to cleave ADAMTS-18. The generated ~45-kDa C-terminal cleavage product of ADAMTS-18 becomes activated. It clusters the β3 integrins and induces oxidative platelet fragmentation as we previously described anti-GPIIIa 49 - 66 Ab [17] . We have identified that R⁷⁷⁵/S ^{77 6} in spacer region of ADAMTS-18 is one of the potential cleavage sites of thrombin [18] . However, sequence analysis indicates that there still exist two same sites in spacer region neighboring R⁷⁷⁵/S⁷⁷⁶ named R⁷⁹²/S⁷⁹³ and R⁸²³/S⁸²⁴, which also generate similar ~45-kDa C-terminal products in theory when cleaved by thrombin. In this study, we have investigated other thrombin cleavage sites in spacer region of ADAMTS-18 through amino acid mutation and mass spectrometry assay.

Despite the similarity shared by ADAMTS family members, most differences among them are found in the C- terminal domains of the protein, suggesting that the C-terminal domains of ADAMTS may determine their in vivo location and substrate specificity [19] - [22] . C-terminal processing has been shown in ADAMTS-1 [19] [21] , ADAMTS-4 [20] [22] , ADAMTS-8 [4] , ADAMTS-9 [5] , and ADAMTS-13 [6] . This splicing will shed light on the biological function of these important proteins. Noteworthily, most of cleavage events occur within the spacer region [19] - [22] .

We previously reported that cleavage of ADAMTS-18 by thrombin represent a novel mechanism for platelet thrombus clearance [17] . The release of the active 45-kDa C-terminal fragment could regulate thrombus size by inducing oxidative platelet fragmentation. In this study, we first revealed that the R⁷⁷⁵/S⁷⁷⁶, R⁷⁹²/S⁷⁹³ and R⁸²³/S⁸²⁴ in spacer region of ADAMTS-18 are critical for thrombin cleavage. This cleavage region is similar to those of the ADAMTS family members reported previously [19] - [22] . Physiologically, thrombin is generated rapidly, and at high local concentrations during the normal hemostatic response. We found that ADAMTS-18 was proteolyzed by thrombin at a high thrombin concentration, whereas low thrombin concentration had undetectable cleavage effect on ADAMTS-18 which mimics some physiological conditions, especially platelet thrombus formation. It is of interest in this regard that ADAMTS-13 has recently been shown to be inactivated by thrombin contributing to the loss of ADAMTS-13 VWF cleavage function [6] . Furthermore, ADAMTS-13 has been reported to limit platelet thrombus formation in a shear rate dependent platelet thrombus model on collagen, by its cleavage of ultra large VWF [23] .

In present study, we also find ~75-kDa band which is from ADAMTS-18 cDNA in in vitro translation assays. Thrombin or other enzyme (cathepsin D or trypsin) had no effect on the generation of this band. Thus, it is likely other mechanism has been involved in the ADAMTS-18 processing.

In summary, this report provides a direct proof that the existence of C-terminal proteolytic cleavage sites of ADAMTS-18 by thrombin, has potential drug application in dissolution of arterial thrombi.

This work was supported by the National Natural Science Foundation of China (No.81170481, 81200352); the Innovation Fund of Shanghai Municipal Education Commission (12zz040, 13YZ024), Shanghai Municipal Natural Science Foundation (12ZR1421100), The Key Construction Program of the National “ 985” project, and SRF for ROCS (to W.Z.), Doctoral Fund of Ministry of Education of China (20120073120113).