Mechanism of Factor Va Inactivation by Plasmin

Abstract

The coagulation cofactor Va (FVa) is a noncovalent heterodimer consisting of a heavy chain (FVaH) and a light chain (FVaL). Previously, the fibrinolytic effector plasmin (Pn) has been shown to inhibit FVa function. To understand this mechanism, the fragmentation profile of human FVa by Pn and the noncovalent association of the derived fragments were determined in the presence of Ca 2؉ using anionic phospholipid (aPL)-coated microtiter wells and large (1 m) aPL micelles as affinity matrices. Following Pn inactivation of aPL-bound FVa, a total of 16 fragments were observed and their NH 2 termini sequenced. These had apparent molecular weights and starting residues as follows (single letter abbreviation is used): 50(L1766), 48(L1766), 43(Q1828), 40(Q1828), 30(S1546), 12(T1657), and 7(S1546) kDa from

Retinoschisin (RS1), the Protein Encoded by the X-linked Retinoschisis Gene, Is Anchored to the Surface of Retinal Photoreceptor and Bipolar Cells through Its Interactions with a Na/K ATPase-SARM1 Complex

“…Synthetic phospholipids (3:1 ratio of DOPC to DOPS, 3:1 ratio of DOPC to DOPE or 1:1:1 ratio of DOPC:DOPS:DOPE) were dissolved in a 1:1 mixture of chloroform:methanol and dried to a homogeneous film under a stream of nitrogen gas. The synthetic phospholipids or retinal lipids were extracted with 20 mM HEPES buffer, pH 7.4, containing 300 mM sucrose and extensively vortexed (27). The resulting multilamellar vesicles were subjected to 5 freeze-thaw cycles in liquid nitrogen to promote lipid hydration and equal transmembrane lipid distribution.…”

Residues 110–126 in the A1 Domain of Factor VIII Contain a Ca2+ Binding Site Required for Cofactor Activity

“…Furthermore, the observation that Ca 2ϩ binding to A2 domain in HC contributes little if at all to generate cofactor activity highlights the functional role of the Ca 2ϩ binding site in A1 domain in HC. Recently, Zeibdawi et al (22) reported that residues 94 -110 in factor V comprise a Ca 2ϩ binding site required for its activity. In the current study, we probed the homologous region in the A1 domain of factor VIII (residues 110 -126) for Ca 2ϩ binding using a site-directed mutagenesis approach.…”

“…Although Ca 2ϩ binding motifs have been identified, many Ca 2ϩ -binding proteins coordinate the ion at sites other than a consensus sequence. Recently it was reported that residues 94 -110 in factor V, a region rich in acidic amino acids, bound Ca 2ϩ , promoting the association of factor Va HC and LC (22). An homologous region in factor VIII, residues 110 -126, contains 4 Asp and 4 Glu residues in the 16-residue segment.…”

Does plasmin have anticoagulant activity?

“…FVa is inactivated by plasmin cleavage in both heavy and light chains 13,20. Plasmin cleaves the heavy chain of FV at three sites, Lys309, Lys310, and Arg313, releasing the A 2 domain and causing inactivation of the cofactor 21,22. The plasmin cleavages are accelerated in the presence of a membrane surface 21.…”

“…The plasmin cleavages are accelerated in the presence of a membrane surface 21. Measuring thrombin formation20,22 or plasma clotting21 determines the functional activity of FV. In humans, a deficiency of FV causes excessive bleeding.…”