Plasmid construction

All CTLD 14 members, mutants and chimeras inserted between EcoRI in pEGFPN1, using Gibson assembly according to manufacturer (New England Biolabs, Hitchin, UK), using PCR products amplified with the following primers; CLEC14A-forward 5′-GATCTCGAGCTCAAGCTTCGATGAGGCCGGCGTTCGCC-3′, CLEC14A-reverse 5′-TACCGTCGACTGCAGTGCATCACTAGAGCCAAG-3′, CD93-forward 5′-CGAGCTCAAGCTTCGATGGCCACCTCCATGGGC-3′, CD93-reverse 5′-TACCGTCGACTGCAGGCAGTCTGTCCCAGGTGTCG-3′, THBD-forward 5′-CGAGCTCAAGCTTCGATGCTTGGGGTCCTGGTC-3′, THBD-reverse 5′-TACCGTCGACTGCAGGAGTCTCTGCGGCGTCCG-3′, CD248-forward 5′-CGAGCTCAAGCTTCGATGCTGCTGCGCCTGTTG-3′, CD248-reverse 5′-TACCGTCGACTGCAGTCACACGCTGGTTCTGCAG-3′. For chimeras, two PCR products or more were Gibson assembled using; (CLEC14A^THBD(CTLD); THBD-forward and THBD-CTLD fused to CLEC14A-sushi-reverse 5′-CTCAAACTGGAACTCGCAGAGGAAGCC-3′, THBD-CTLD fused to CLEC14A-sushi-forward 5′-GCGAGTTCCAGTTTGAGGTCTTGTGTC-3′ and CLEC14A-reverse). (CLEC14A^THBD(sushi); CLEC14A-forward and CLEC14A-CTLD fused to THBD-sushi-reverse 5′-TACCGTCGACTGCAGTGCATCACTAGAGCCAAG-3′, CLEC14A-CTLD fused to THBD-sushi-forward 5′-GTGCAAGTACCACTTCCCAGCCACCTGCAGGC-3′ and THBD-sushi fused to CLEC14A-EGF-reverse 5′-TCCCGGGGCAAGCGCCCGGCGCCTCCCT-3′, THBD-sushi fused to CLEC14A-EGF-forward 5′-GCCGGGCGCTTGCCCCGGGAGGTACCTC-3′ and CLEC14A-reverse). (CLEC14A^C103S; CLEC14A-forward and CLEC14A^C103S-reverse 5′-CTCGTTCTCCAGGGTTGAGTGGGAACGCCTGCGCTC-3′, CLEC14A^C103S-forward 5′-GAGCGCAGGCGTTCCCACTCAACCCTGGAGAACGAG-3′ and CLEC14A-reverse). (CLEC14A^C138S; CLEC14A-forward and CLEC14A^C138S-reverse 5′-CGCGCATCTCCGCGCGGTGGAGGAGCGTTGGGGCTCCTC-3′, CLEC14A^C138S-forward 5′-GAGGAGCCCCAACGCTCCTCCACCGCGCGGAGATGCGCG-3′ and CLEC14A-reverse). (CLEC14A^CD248(sushi); CLEC14A-forward and CLEC14A-CTLD fused to CD248-sushi-reverse 5′-CCTCGAAGCCGTACTTGCACAGGTAGCCGTTGGC-3′, CLEC14A-CTLD fused to CD248-sushi-forward 5′-GTGCAAGTACGGCTTCGAGGGCGCCTGC-3′ and CD248-sushi fused to CLEC14A-EGF-reverse 5′-TCCCGGGGCAGCCAGTCCCCAGGCACAGG-3′, CD248-sushi fused to CLEC14A-EGF-forward 5′-GGGGACTGGCTGCCCCGGGAGGTACCTC-3′ and CLEC14A-reverse). (CLEC14A^CD248(CTLD); CD248-forward and CD248-CTLD fused to CLEC14A-sushi-reverse 5′-CTCAAACTGAAACTGGCACAGGTAGCCG-3′, CD248-CTLD fused to CLEC14A-sushi-forward 5′-GCCAGTTTCAGTTTGAGGTCTTGTGTC-3′ and CLEC14A-reverse).

MMRN2 fragments were amplified using; (MMRN2^EMI-CC; MMRN2^FL-forward 5′-CCGGACCGGTCAGGCTTCCAGTACTAGCC-3′ and MMRN2⁸²⁰-reverse 5′-CTACTAGGTACCCCAGAGCGCCGCGCCC-3′). (MMRN2^CC-C1q; MMRN2¹³³-forward 5′-CCGGACCGGTGATTCCATGGCAATCCCTGA-3′ and MMRN2^FL-reverse 5′-CGGGGTACCGGTCTTAAACATCAGGAAGC-3′). (MMRN2^CC; MMRN2¹³³-forward and MMRN2⁸²⁰-reverse). (MMRN2^133–486; MMRN2¹³³-forward and MMRN2⁴⁸⁶-reverse 5′-CTACTAGGTACCCTTGATGAGGTCGGCATGG-3′). (MMRN2^487–820; MMRN2⁴⁸⁷-forward 5′-CCGGACCGGTTACGTGAAGGACTGCAATTG-3′ and MMRN2⁸²⁰-reverse), (MMRN2^487–674; MMRN2⁴⁸⁷-forward and MMRN2⁶⁷⁴-reverse 5′-CTACTAGGTACCCGGCCGCGGGGGCTCCG-3′) (MMRN2^675–820; MMRN2⁶⁷⁵-forward 5′-CCGGACCGGTGCAGAGCACCTGGAGCC-3′ and MMRN2⁸²⁰-reverse) (MMRN2^487–603; MMRN2⁴⁸⁷-forward and MMRN2⁶⁰³-reverse 5′-CTACTAGGTACCCGCGTCCTCCAGCAGGG-3′) (MMRN2^604–674; MMRN2⁶⁰⁴-forward 5′-CCGGACCGGTCTGCGGCACGAGGCGGTG-3′ and MMRN2⁶⁷⁴-reverse) (MMRN2^530–624; MMRN2⁵³⁰-forward 5′-CCGGACCGGTGGCTCCTCCCTGCAGGCC-3′ and MMRN2⁶²⁴-reverse 5′-CTACTAGGTACCCTCAGACATCTCCTCCAGC-3′) (MMRN2^495–674; MMRN2⁴⁹⁵-forward 5′-TAGTAGACCGGTCAGAAGCTCTATTTAGACCTG-3′ and MMRN2⁶⁷⁴-reverse). (Mouse MMRN2^495–678; mMMRN2⁴⁹⁵-forward 5′-CCGGACCGGTCAAAGGGTCAACTCTGACGTG-3′ and mMMRN2⁶⁷⁸-reverse 5′-CTACTAGGTACCCAACTGTGGGTGCTGCTCC-3′). AgeI and KpnI digested PCR products were ligated into pHL-Avitag3 containing; N-terminal signal peptide (SP), C-terminal BirA and His tag (avitag).³⁶

Codon optimized MMRN2^495–674 and MMRN2^495–603 DNA was synthesized (IDT Technologies, Leuven, Belgium) with ends complementary to pET23a vector and Gibson assembled in between NdeI and NotI. The BirA sequence 5′-GGTGGTGGTCTGAACGATATTTTTGAAGCTCAGAAAATCGAATGG-3′ was used.

Lentiviral vectors were Gibson assembled between PmeI sites using primers: mCLEC14A-ECD; forward 5′-ACTAGCCTCGAGGTTTAAACATGAGGCCAGCGCTTGCC-3′ and reverse 5′-CACTCGATGAGGATCCGGAAGAGGTGTCGAAAGTCAGAGAAAC-3′, mouse Fc for fusion to mCLEC14A-ECD-forward 5′-CCTCTTCCGGATCCTCATCGAGTGTGCCCAGGGATTGTGGT-3′ and reverse 5′-CTGCAGCCCGTAGTTTTCATTTACCAGGAGAGTGGG-3′. mFc alone fused to CLEC14A SP; mCLEC14A SP-forward 5′-AGACTAGCCTCGAGGTTTAAACATGAGGCCAGCGCTTGC-3′ and mCLEC14A SP-reverse 5′-TGAGGATCCCTCCCCATTCCCTGGCCG-3′, mFc fused to SP; forward 5′-AATGGGGAGGGATCCTCATCGAGTGTG-3′ and reverse 5′-TCCTGCAGCCCGTAGTTTTCATTTACCAGGAGAGTGG-3′. mMMRN2^495–678 inserted between engineered BamHI site separating the SP and mFc; forward 5′-CAGGGAATGGGGAGGGATCCCAAAGGGTCAACTCTGACG-3′ and reverse 5′-GGCACACTCGATGAGGATCCCAACTGTGGGTGCTGCTC-3′. Human and mouse MMRN2, human and mouse CLEC14A, CD248 and thrombomodulin amplified from IMAGE clones, mFc amplified from cDNA of C3 hybridomas. CD93 amplified from pCDNA3-CD93, a gift from Suzanne Bohlson. CLEC14A domain deletions are as previous.⁴