Partial Glycosylation at Asparagine-2181 of the Second C-Type Domain of Human Factor V Modulates Assembly of the Prothrombinase Complex

105

Platelet-and plasma-derived factor Va (FVa) serve essential cofactor roles in prothrombinase-catalyzed thrombin generation. Platelet-derived FV/Va, purified from Triton X-100 platelet lysates was composed of a mixture of polypeptides ranging from ϳ40 to 330 kDa, mimicking those visualized by Western blotting of platelet lysates and releasates with anti-FV antibodies. The purified, platelet-derived protein expressed significant cofactor activity such that thrombin activation led to only a 2-3-fold increase in cofactor activity yet expression of a specific activity identical to that of purified, plasma-derived FVa. Physical and functional differences between the two cofactors were identified. Purified, platelet-derived FVa was 2-3-fold more resistant to activated protein C-catalyzed inactivation than purified plasma-derived FVa on the thrombin-activated platelet surface. The heavy chain subunit of purified, plateletderived FVa contained only a fraction (ϳ10 -15%) of the intrinsic phosphoserine present in the plasma-derived FVa heavy chain and was resistant to phosphorylation at Ser 692 catalyzed by either casein kinase II or thrombin-activated platelets. MALDI-TOF mass spectrometric analyses of tryptic digests of platelet-derived FV peptides detected an intact heavy chain uniquely modified on Thr 402 with an N-acetylglucosamine or N-acetylgalactosamine, whereas Ser 692 remained unmodified. N-terminal sequencing and MALDI-TOF analyses of plateletderived FV/Va peptides identified the presence of a fulllength heavy chain subunit, as well as a light chain subunit formed by cleavage at Tyr 1543 rather than Arg 1545 accounting for the intrinsic levels of cofactor activity exhibited by native platelet-derived FVa. These collective data are the first to demonstrate physical differences between the two FV cofactor pools and support the hypothesis that, subsequent to its endocytosis by megakaryocytes, FV is modified to yield a platelet-derived cofactor distinct from its plasma counterpart.Factor Va (FVa), 1 a heterodimeric protein composed of heavy chain (105 kDa) and light chain (74 kDa) subunits, is formed by limited proteolysis of factor V (FV) (1). In normal hemostasis, FVa functions as a non-enzymatic cofactor of the prothrombinase complex, which consists of a 1:1 stoichiometric and Ca 2ϩ -dependent complex of the serine protease factor Xa and FVa, bound to the membrane of appropriately activated platelets, and catalyzes the proteolytic conversion of prothrombin to thrombin (2). When incorporated into the prothrombinase complex, the catalytic activity of factor Xa is increased by approximately 5 orders of magnitude, and FVa contributes substantially to this increase (3). Removal of FVa from the prothrombinase complex results in a 10,000-fold decrease in the rate of thrombin generation (3), the physiologic effect of which is demonstrated in the bleeding diatheses expressed by FV-deficient individuals (4 -8)Factor V circulates in two pools in whole blood. The majority (75-80%) is found in the plasma as an inactive, singl...

Section: Platelet-derived Fv/va Is Specifically Modified By the Additmentioning

confidence: 72%

Unique in Vivo Modifications of Coagulation Factor V Produce a Physically and Functionally Distinct Platelet-derived Cofactor

Gould¹,

Silveira²,

Tracy

2004

105

“…We assumed that the change in intrinsic fluorescence should be proportional to the fraction of protein bound and fitted the titration data using a model that assumed four identical and independent sites (10). We have shown that there is a 3-fold difference in the affinity of both human (12) and bovine (14) FV a1 and FV a2 for PS-containing membranes. Unlike their interactions with membranes, we conclude that bovine FV a1 and FV a2 bind to soluble C6PS with comparable affinities (11 Ϯ FIG.…”

Section: Effect Of C6ps On Prothrombin Activation By Fx a In Thementioning

confidence: 99%

“…This has been attributed to substantially different affinities for binding to membranes (13). However, our lab has reported that the two forms of both bovine and human FV a bind to membranes with only ϳ3-fold different affinities (12,14). This suggests that differences in the ability to support prothrombinase activity must reflect either different binding between factors FX a and FV a1 versus FV a2 or different intrinsic activities of the FX a ⅐FV a1 and FX a ⅐FV a2 complexes.…”

mentioning

confidence: 98%

See 1 more Smart Citation

Soluble Phosphatidylserine Triggers Assembly in Solution of a Prothrombin-activating Complex in the Absence of a Membrane Surface

Majumder

Weinreb

Zhai

et al. 2002

Self Cite

Factor X a (FX a ) binding to factor V a (FV a ) on plateletderived membranes containing surface-exposed phosphatidylserine (PS) forms the "prothrombinase complex" that is essential for efficient thrombin generation during blood coagulation. There are two naturally occurring isoforms of FV a , FV a1 and FV a2 . These two isoforms differ by a 3-kDa polysaccharide chain ( ). The ability of soluble PS to trigger formation of a soluble prothrombinase complex suggests that exposure of PS molecules during platelet activation is likely the key event responsible for the assembly of an active membrane-bound complex.The final step in the blood coagulation cascade involves the activation of prothrombin to thrombin, which is the central enzyme of the coagulation system. This activation requires assembly of an enzyme complex, called prothrombinase (1), which consists of blood coagulation factors X a (a serine protease) and V a (a cofactor), Ca 2ϩ , and membranous vesicles derived from stimulated platelets (2). Several studies (3-5) have suggested that phosphatidylserine (PS) 1 might play a specific role in prothrombin activation. PS is asymmetrically distributed to the cytoplasmic surface of resting platelet membranes (6) but is exposed when human platelets are activated (7). It has become clear only very recently that PS regulates the structure and function of factors X a and V a (8, 10). 2 Here we explore further the extent of this regulation.Factor V exists in plasma as an inactive, single chain glycoprotein with a molecular mass of 330 kDa. The active form of factor V, FV a , has a central domain removed to yield a heterodimer composed of two chains, a heavy chain (M r ϭ 94,000 in the bovine species; 105,000 in human) and a heterogeneous light chain (M r ϭ 74,000 in FV a1 or 71,000 in FV a2 ). The heavy and light chains form a tight complex in the presence of a calcium ion (11). The heterogeneity in the light chain is seen in both the bovine and human molecules. In the human form, it appears to arise from glycosylation of Asn 2181 at the C-terminal end of the light chain (12). Prothrombinase complexes assembled from the two molecular species derived from human plasma are observed to have somewhat different cofactor activities (13,14). This has been attributed to substantially different affinities for binding to membranes (13). However, our lab has reported that the two forms of both bovine and human FV a bind to membranes with only ϳ3-fold different affinities (12,14). This suggests that differences in the ability to support prothrombinase activity must reflect either different binding between factors FX a and FV a1 versus FV a2 or different intrinsic activities of the FX a ⅐FV a1 and FX a ⅐FV a2 complexes. Although our results have favored the former possibility (14), it has been difficult to prove this unambiguously because it is difficult to measure precisely the interaction between FX a and FV a on a membrane surface (15).The presence of FV a in a reaction mixture is critical to obtaining a maximal and physiologicall...

“…However, factor VIII requires more PS per binding site than factor V (13), and current evidence implicates different domains in membrane binding. While binding of factor V is apparently mediated by both the A3 domain (17) and the C2 domain (18) and is influenced by glycosylation within the C2 domain (19), binding of factor VIII is apparently mediated by the C2 domain (20,21) and is independent of glycosylation. Recent publication of two crystal structures for the C2 domain of factor V (22) and a single structure for the C2 domain of factor VIII (23) has provided the basis for a model membrane-binding mechanism.…”

mentioning

confidence: 99%

Four Hydrophobic Amino Acids of the Factor VIII C2 Domain Are Constituents of Both the Membrane-binding and von Willebrand Factor-binding Motifs

Gilbert¹,

Kaufman²,

Arena³

et al. 2002

111

148

Factor VIII binds to phospholipid membranes and to von Willebrand factor (vWf) via its second C domain, which has lectin homology. The crystal structure of the C2 domain has prompted a model in which membrane binding is mediated by two hydrophobic spikes, each composed of a pair of residues displayed on a ␤-hairpin turn, and also by net positive charge and specific interactions with phospho-L-serine. , and 91% reduction in specific activity in the activated partial thromboplastin time assay. In a phospholipidlimiting factor Xa activation assay, these mutants had a 65, 85, and 96% reduction in specific activity. Equilibrium binding of fluorescent, sonicated phospholipid vesicles to mutants immobilized on Superose beads was measured by flow cytometry. The affinities for phospholipid were reduced ϳ20-, 30-, and >35-fold for 2199/2200, 2251/2252, and 4-Ala, respectively. A dimeric form of mature vWf bound to immobilized factor VIII and the same mutants, but the affinities of the mutants were reduced ϳ5-, 10-, and >20-fold, respectively. In a competition, solution phase enzyme-linked immunosorbent assay, plasma vWf bound factor VIII and the same mutants with the affinities for the mutants reduced >5-, >5-, and >50-fold, respectively. We conclude that the two hydrophobic spikes are constituents of both the phospholipidbinding and vWf-binding motifs. In plasma, vWf apparently binds the inherently sticky membrane-binding motif, preventing nonspecific interactions.Factor VIII is a phosphatidyl-L-serine (PS) 1 binding cofactor (1, 2) for the vitamin K-dependent serine protease, factor IXa, that also binds to PS-containing membranes (3, 4). The membrane-bound factor VIIIa-factor IXa complex cleaves the zymogen, factor X, to factor Xa, which is then responsible for catalyzing prothrombin activation (5). The importance of this enzyme complex is illustrated by hemophilia, a disease in which a deficiency of either factor VIII (hemophilia A) or factor IX (hemophilia B) leads to life-threatening bleeding. Factor IXa gains more than 100,000-fold greater efficiency in activating factor X by assembling with factor VIIIa on a PS-containing membrane than when free in solution (6). We have recently found that the predominant effect of PS-containing membranes on the factor VIIIa-factor IXa complex is to increase the k cat by more than 1000-fold (7). These membranes also increase the affinity of factor IXa for factor VIIIa and for factor X. The central importance of the membrane binding function of factor VIII motivates studies to define the membrane-binding motif.Factor VIII, with M r 280,000, is homologous to another procoagulant protein, factor V, in amino acid sequence (8 -10) and in function, as a membrane-bound enzyme cofactor (5, 11-13). The proteins share a repeating domain structure of A1-A2-B-A3-C1-C2 in which the A domains are homologous with ceruloplasmin, the B domain is unique to each protein, and the C domains are homologous with discoidin I, a phospholipid-binding lectin (14), and with a murine milk fat globule membrane ...