Characterization of a thrombomodulin binding site on protein C and its comparison to an activated protein C binding site for factor Va

Abstract

Activation of the anticoagulant human plasma serine protease zymogen, protein C, by a complex of thrombin and the membrane protein, thrombomodulin, generates activated protein C, a physiologic anti-thrombotic, anti-inflammatory and anti-apoptotic agent. Alanine-scanning site-directed mutagenesis of residues in five surface loops of an extensive basic surface on protein C was used to identify residues that play essential roles in its activation by the thrombin-thrombomodulin complex. Twenty-three residues in the protein C protease domain were mutated to alanine, singly, in pairs or in triple mutation combinations, and mutants were characterized for their effectiveness as substrates of the thrombin-thrombomodulin complex. Three protein C residues, K192, R229, and R230, in two loops, were identified that provided major contributions to interactions with thrombin-thrombomodulin, while six residues, S190, K191, K217, K218, W231, and R312, in four loops, appeared to provide minor contributions. These protein C residues delineated a positively charged area on the molecule's surface that largely overlapped the previously characterized factor Va binding site on activated protein C. Thus, the extensive basic surface of protein C and activated protein C provides distinctly different, though significantly overlapping, binding sites for recognition by thrombin-thrombomodulin and factor Va.

The cytoprotective protein C pathway

“…The anticoagulant action of APC critically involves a cleavage site at Arg506 in factor Va, and this cleavage depends on certain positively charged residues in surface loops on APC's protease domain, including loop 37 (protein C residues 190-193, equivalent to chymotrypsin [CHT] residues 36-39), the Ca ϩϩ -binding loop (residues 225-235, CHT residues 70-80), and the autolysis loop (residues 301-316, CHT residues 142-153) ( Figure 5A). 112,[114][115][116][117][118][119] Two APC variants were made with Ala mutations in 2 surface loops and involved Ala replacements of Arg229 and Arg230 and of Lys191, Lys192, and Lys193 ( Figures 5A,C). Each APC variant had little anticoagulant activity (4% to 14% compared with wild-type APC) (Figure 5B), whereas each APC variant retained normal antiapoptotic activity, requiring PAR-1 and EPCR ( Figure 5D).…”

Regulation of Blood Coagulation by the Protein C Anticoagulant Pathway

“…7,19,20 The EGF domains play a crucial role in the activation of protein C, thrombin binding to EGF5 and EGF6, whereas EGF4 interacts with a positively charged cluster formed by basic residues located in loops 37, 60, 70, and 148 (minor role) in the SP domain of protein C (Figure 2). 7,19,[21][22][23][24][25] EPCR augments the activation of protein C by binding the Gla domain of protein C, thereby aligning the substrate protein C with the activating T-TM complex. 7,14 EPCR is a type I membrane protein and a member of the MHC class 1/CD1 family.…”

Peran Trombomodulin untuk Mendeteksi Kerusakan Sel Endotel pada Malaria Tropika dan Malaria Tertiana

“…21,22 Trombomodulin adalah reseptor permukaan sel endotel untuk trombin, fungsi TM mengubah protein C yang terikat pada trombin menjadi aktivator protein C. Setelah protein C teraktivasi, maka TM bekerja sebagai antikoagulan utama lewat kemampuannya menginaktivasi berbagai faktor pembekuan (Va, VIIIa, Xa dan XIIIa). Aktivasi protein C merupakan hal yang sangat penting dalam meregulasi proses koagulasi dan reaksi inflamasi.…”

“…Sebagian reseptor di permukaan sel endotel ialah trombomodulin (TM), thrombospondin (TSP), CD 36 , ICAM-1, E-selectin, VCAM-1, PECAM-1, P-selectint berinteraksi dengan eritrosit yang terinfeksi parasit (PRBC) melalui PECAM-1. 6,13,21,22 Ohnishi dkk, 3 melakukan penelitian di Jepang pada 6 pasien malaria tertiana dengan menghubungkan serum TM, ICAM-1, VCAM-1, E-selectin. Terdapat hubungan yang antara serum TM antara kelompok malaria tertiana dengan kelompok kontrol (p<0,005).…”

“…Aktivasi dari sel endotel yang diakibatkan oleh adhesi eritrosit yang terinfeksi mengakibatkan TM berlebihan keluar dari permukaan sel endotel. 6,13,21,22 Pada penelitian ini, hasil uji t menunjukkan perbedaan kadar TM untuk mendeteksi kerusakan sel endotel antara malaria tropika dengan komplikasi dan tanpa komplikasi (p=0,009). Penelitian ini hampir sama hasilnya dengan penelitian yang dilakukan Rogerson dkk, 9 di Malawi pada 155 anak.…”