Although python27.exe.lnk is callable from any command line tool, with any of its arguments the subprocess module thinks it's not a valid Win32 application.

Proof, let there be python27.exe.lnk be a shortcut to python.exe:

C:\Python27
λ ls python*
python.exe*  python27.exe.lnk*  pythonw.exe*

λ python.exe -c "import sys; print(sys.version)"
2.7.13 (v2.7.13:a06454b1afa1, Dec 17 2016, 20:42:59) [MSC v.1500 32 bit (Intel)]

C:\Python27
λ python.exe -c "import subprocess; print(subprocess.call(['./python.exe', '-c', '\"import sys; print(sys.version)\"']))"
0

C:\Python27
λ python27.exe -c "import subprocess; print(subprocess.call(['./python27.exe.lnk', '-c', '\"import sys; print(sys.version)\"']))"
Traceback (most recent call last):
  File "<string>", line 1, in <module>
  File "C:\Python27\lib\subprocess.py", line 168, in call
    return Popen(*popenargs, **kwargs).wait()
  File "C:\Python27\lib\subprocess.py", line 390, in __init__
    errread, errwrite)
  File "C:\Python27\lib\subprocess.py", line 640, in _execute_child
    startupinfo)
WindowsError: [Error 193] %1 is not a valid Win32 application