The factor Xa-catalyzed activation of factor V.

494

467

Human factor VIII was isolated from commercial factor VIII concentrates and found to consist of multiple polypeptides with molecular weights ranging from 80 000 to 210 000. Immunological and amino acid sequence data identified these polypeptides as subunits of factor VIII. N-Terminal amino acid sequence analysis determined that the Mr 210 000 and 80 000 proteins are derived from the N- and C-terminal portions of factor VIII, respectively; Mr 90 000-180 000 polypeptides are derived from the Mr 210 000 polypeptide by C-terminal cleavages. Treatment of purified factor VIII with thrombin resulted in proteolysis of Mr 80 000-210 000 proteins and the generation of polypeptides of Mr 73 000, 50 000, and 43 000. Maximum coagulant activity of thrombin-activated factor VIII was correlated with the generation of these polypeptides. The proteolysis as well as activation of factor VIII by thrombin was found to be markedly dependent on CaCl2 concentration. Proteolysis of factor VIII with activated protein C (APC) resulted in degradation of the Mr 90 000-210 000 proteins with the generation of an Mr 45 000 fragment. This cleavage correlated with inactivation of factor VIII by APC. The Mr 80 000 protein was not degraded by APC. Factor Xa cleaved the Mr 80 000-210 000 factor VIII proteins, resulting in the generation of fragments of Mr 73 000, 67 000, 50 000, 45 000, and 43 000. Factor Xa was found to initially activate and subsequently inactivate factor VIII.(ABSTRACT TRUNCATED AT 250 WORDS)

Section: Discussionmentioning

confidence: 99%

Proteolytic processing of human factor VIII. Correlation of specific cleavages by thrombin, factor Xa, and activated protein C with activation and inactivation of factor VIII coagulant activity

Eaton¹,

Rodriguez²,

Vehar³

1986

494

467

“…The derived amino acid sequences correspond to a molecule with triplicated "A" domains, duplicated "C" domains, and a "B" region. Factor V is cleaved by thrombin and factor Xa to generate the active cofactor, factor Va (Nesheim & Mann, 1979;Foster et al, 1983b;Monkovic & Tracy, 1991). Bovine factor Va is composed of a heavy chain (A1-A2 domains, factor Vanc> Mr = 94 000) derived from the NH2-terminal portion of the factor V molecule and a light chain (A3-C1-C2, factor VaLc, Mr = 74 000) derived from the COOH-terminal domain of factor V (Nesheim & Mann, 1979, Guinto et al, 1992.…”

mentioning

confidence: 99%

Contribution of the Heavy and Light Chains of Factor Va to the Interaction with Factor Xa

Kalafatis¹,

Xue²,

Lawler³

et al. 1994

The interactions of the isolated heavy and light chains of factor Va with factor Xa were evaluated using active-site-modified factor Xa [(carboxytetramethyl)rhodamine-Glu-Gly- Arg-factor Xa (ctr-EGR-Xa)]. The Kd for the factor Va heavy-chain interaction with ctr-EGR-Xa was 60 microM. A series of monoclonal antibodies directed against bovine factor Va were tested for their ability to inhibit thrombin formation in an assay using the fluorescent thrombin inhibitor dansylarginine N,N-(3-ethyl-1,5-pentanediyl)amide (DAPA). Monoclonal antibody alpha BFV-4, which recognizes the light chain of the cofactor, was found to inhibit the formation of thrombin. Similarly, monoclonal antibody alpha BFV-5, which is directed against the heavy chain of the cofactor, was found to inhibit thrombin formation. In contrast, monoclonal antibody alpha BFV-1, also directed against the heavy chain of the cofactor, did not inhibit thrombin generation by the prothrombinase complex. Monoclonal antibodies alpha BFV-4 and alpha BFV-5 inhibited the interaction of active-site-modified radiolabeled factor Xa (125I-Xa-EGR) with factor Va bound to PC/PS-coated microtiter wells, whereas nonimmune mouse IgG did not have any effect on the 125I-Xa-EGR.membrane-bound factor Va interaction. The antibodies effect upon the phospholipid-independent interaction between the cofactor and ctr-EGR-Xa was evaluated by analytical ultracentrifugation. Both alpha BFV-4 and alpha BFV-5 inhibited the phospholipid-independent interaction between factor Va and ctr-EGR-Xa.(ABSTRACT TRUNCATED AT 250 WORDS)

“…The C domains contain 2 "7" loops of 154 (Cl) and 155 (C2) residues, respectively, 1866-2020 and 2025-2180. Factor V is an Mr = 330 000 single-chain glycoprotein present in plasma and platelets which is an essential component of the blood coagulation cascade (Owren, 1947;Mann et al, 1981Mann et al, , 1988; Kane & Davie, 1988). Factor V is activated to its active form, factor Va, by thrombin and factor Xa Nesheim et al, 1984;Suzuki et al, 1982;Foster et al, 1983; Monkovic & Tracy, 1990). Factor Va serves as a cofactor in the prothrombinase complex which catalyzes the activation of prothrombin to thrombin (Esmon, 1979; Krishnaswamy et al, 1989Krishnaswamy et al, , 1993).…”

mentioning

confidence: 99%

Determination of the disulfide bridges in factor Va heavy chain

Xue

Kalafatis²,

Silveira³

et al. 1994

The M(r) = 94,000 heavy chain of bovine factor Va contains 10 cysteine residues which are distributed in the 2 A domains which make up this portion of the factor V molecule. The A1 domain contains four cysteines while the A2 domain contains six cysteines. The locations of disulfide bridges and free cysteines in bovine factor Va heavy chain were analyzed using iodo[14C]acetamide-labeled factor Va heavy chain digested with trypsin, plasmin, V-8 protease, and cyanogen bromide. Following HPLC separation of the resulting peptides, free cysteines were identified by the incorporation of radioactivity while disulfide-containing peptides were detected using an SBD-F fluorometric assay after reduction. All cysteine-containing peptides were analyzed by amino acid sequence analysis. The four cysteines in the A1 domain are associated with two disulfide bonds, Cys139-Cys165 and Cys220-Cys301. One disulfide bond was explicitly identified in the A2 domain; Cys471-Cys497, and a free cysteine was found in the A2 domain at Cys538. Significant difficulties were encountered in preparing identifiable or soluble peptides which would permit the explicit identification of the three remaining cysteines in the A2 domain. On the basis of homology, it is likely that Cys589 is a free SH while a disulfide bridge exists between Cys579 and Cys660. Thus, three major disulfide bonding patterns, characterized as "alpha", "beta", and "gamma" loops, are found in factor V. Each A domain contains a 26 residue "alpha loop at positions 139-165, 471-497, and 1684-1710. The A1 and A2 domains each contain 81 amino acid residue "beta" loops at 220-301 and 579-660.(ABSTRACT TRUNCATED AT 250 WORDS)