The Mutations SER2117→LEU, and TRP/TRP2063/64→MET/PHE in the Hydrophobic Spikes of the Factor V C2 Domain Lead to Tenacious Phospholipid Binding and Suggest a Mechanism through Which Phosphatidylserine Activates the Prothrombinase Complex.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2703-2703
Author(s):  
Gary E. Gilbert ◽  
Valerie A. Novakovic ◽  
Randal J. Kaufman ◽  
Hongzhi Miao ◽  
Steven W. Pipe
Keyword(s):  
Specific Activity ◽  
Phospholipid Membranes ◽  
Unexpected Result ◽  
Factor V ◽  
C2 Domain ◽  
Wild Type ◽  
Hydrophobic Residues ◽  
Phospholipid Binding ◽  
Prothrombinase Complex ◽  
Type Factor

Abstract Factor V (fV) binds to phospholipid (PL) membranes via a motif localized to the C2 domain. We and others have shown that PL binding is mediated by two pairs of hydrophobic residues, each displayed at the tips of β-hairpin turns. The homologous hydrophobic residues in the C2 domain of factor VIII also contribute to PL binding. We hypothesized that the solvent-exposed hydrophobic residues of the fV C2 domain make specific contacts that influence membrane affinity and activity of fV. To test this hypothesis we have prepared fVIII/fV hybrid mutants in which amino acid(s) of the fV C2 domain were changed to the homologous residues of fVIII (Mutants #1 W/W 2063/2064 M/F, #2 L/S 2116/2117 L/L, and #3 W/W/S 2063/2064/2117 M/F/L). Mutants were expressed in COS-1 cells and purified by FPLC. The specific activity of the fV/FVIII hybrids #2 and #3 exceeded those of wild type factor V by approx. 10-fold and approx. 4-fold, respectively, in a prothrombin time assay with factor V deficient plasma. Apparent PL affinites were evaluated in a prothrombinase complex assay with limiting phospholipid. The apparent affinities are 9.8 and 21-fold higher than wild type factor V for mutants #2 and #3 which contain the S→L change in the second hydrophobic spike. An unexpected result was that mutants 1–3 supported prothrombinase activity in the absence of added phospholipid, in contrast to wild type fV. We hypothesized that this activity resulted from phospholipid that was not dissociated from fV during purification. This hypothesis was supported because activity was eliminated by incubation of the factor V mutants with phospholipase A2 or by incubation with lactadherin. The tenacity of the phospholipid binding was further investigated by washing immobilized wild type fV and fV mutants with CHAPS prior to elution from an FPLC column. fV mutants #2 and #3 retained activity after the CHAPS wash, free of added PL and activity remained inhibitable by lactadherin, further illustrating the tenacious phospholipid affinity. We utilized Mutants #1 & 2 in the absence of added PL to evaluate the mechanism through which soluble phosphatidylserine with 6-carbon acyl chains (C6PS) enhances activity of the prothrombinase complex. In the absence of phospholipid vesicles C6PS enhanced activity of Mutant 1 equivalent to wild type fV (> 20 fold). However, enhancement of mutant 2 activity, which presumably retained phospholipid via residues 2116/2117, was < 3-fold. This suggests that C6PS functions to activate wild type fV, in part, by engaging the free LS 2116/2117 hydrophobic spike. Together, these data indicate that the hydrophobic spikes of factor V influence the specific activity of factor V, that the high affinity reversible binding of fV to phospholipid membranes is readily perturbed by mutations, and that activation of the prothrombinase complex by C6PS and phospholipid membranes likely involves engagement of amino acids 2116/2117.

His2315/Gln2316 of the Factor VIII C2 Domain Interact with Phospholipid Membranes and Influence Activity of the Factor Xase Complex.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1727-1727
Author(s):  
Gary E. Gilbert ◽  
Randall J. Kaufman ◽  
Patricia Price ◽  
Hongzhi Mao ◽  
Steven W. Pipe
Keyword(s):  
Amino Acids ◽  
Factor Viii ◽  
Phospholipid Bilayer ◽  
Crystallographic Structure ◽  
Phospholipid Membranes ◽  
C2 Domain ◽  
Wild Type ◽  
Phospholipid Binding ◽  
Hydrophobic Amino Acids ◽  
Type Factor

Abstract A major phospholipid-binding motif of factor VIII is localized in the C2 domain. Two pairs of hydrophobic amino acids in the factor VIII C2 domain (Met2199/Phe2200, Leu2251/Leu2252) have previously been shown to interact with phospholipid membranes. However mutations of hydrophilic amino acids (Gln2213, Arg2215, Asn2217, Arg2220, Lys2249) that were predicted to interact with the membrane surface, based upon the crystallographic structure, have not altered phospholipid binding activity. The established hydrophobic interactions do not account for the demonstrated hydrophilic interactions of factor VIII with the phospholipid bilayer. We hypothesized that His2315 and/or Gln2316, interact with phospholipid membranes, based upon their contribution to the epitope for phospholipid-blocking mAb B02C11 and based upon phosphatidylserine binding by C2 domain peptides that include these residues. Factor VIII His2315Ala/Gln2316Ala (fVIII 2315/16) was prepared by PCR mutagenesis and expressed from COS-1 cells. Secreted factor VIII was purified by immunoaffinity chromatography and evaluated in ELISA assays, aPTT assays with factor VIII deficient plasma, and in defined assays with varying phospholipids. The specific activity of fVIII 2315/16 was 67 ± 9% of wild type factor in a commercial aPTT assay with a large excess of phospholipid. The apparent affinity for extruded phospholipid vesicles (0.1 μm diameter) of composition phosphatidylserine:phosphatidylethanolamine: phosphatidylcholine 4:20:76 was 32 ± 10% of wild type factor VIII in a factor Xase assay with varying phospholipid. When the phospholipid concentration was saturating, the Vmax was 55% of factor Xase with wild type factor VIII. We conclude that His2315 and/or Gln2316 constitute the first identified hydrophilic amino acids of factor VIII that interact with a phospholipid membrane. His2315/Gln2316, combined with the two pairs of phospholipid-interactive hydrophobic amino acids constitute three spatially separated phospholipid-interactive points that define a phospholipid-interactive plane on the factor VIII C2 domain. This is consistent with the hypothesis that the phospholipid-binding plane of the C2 domain overlaps substantially with the epitope for BO2C11 and is nearly coincident with the phospholipid-binding surface predicted from the crystallographic structure.

Conservative Mutations in the Membrane-Binding Motif of Factor V C2 Domain to Residues of Pseudonaja Textilis Venom Factor V Confer Phosphatidylserine-Independent Activity

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2243-2243
Author(s):  
Valerie A Novakovic ◽  
Hongzhi Miao ◽  
Steven Pipe ◽  
Gary E. Gilbert
Keyword(s):  
Amino Acids ◽  
Human Factor ◽  
Membrane Binding ◽  
Factor V ◽  
C2 Domain ◽  
Wild Type ◽  
Hydrophobic Membrane ◽  
Prothrombinase Complex ◽  
Membrane Affinity ◽  
Prothrombinase Activity

Abstract Abstract 2243 Toxicity of venom from the eastern brown snake (Pseudonaja textilis) is related to a prothrombin activator protein complex (pseutarin C) that is homologous to the factor Va/factor Xa complex. A previous study has found that the factor V-homologous subunit of this protein (pt-fV) is constitutively active and does not require anionic membranes to function (Bos et al. 2010, Blood). We have previously found that conservative mutation of the amino acids on the hydrophobic membrane binding regions (called spikes) of factor V (W2063M/W2064F/S2117L) can produce increased prothrombinase activity, increased membrane binding affinity and apparent phospholipid-independent prothrombinase activity. However, the membrane-independent activity is caused by retention of phospholipid by factor V through the purification process. We hypothesized that the P.textilis venom-derived factor V has an increase in lipid affinity due to differences in the membrane-interactive spikes. Sequence alignment of the P.textilis venom-derived factor V with bovine and human factor V revealed 5 amino acids located in the putative membrane-binding region (four on spike 3 and one in a region targeted by a small-molecule inhibitor of membrane binding for both factor VIII and factor V) that differed in the venom-derived factor V versus the consensus sequence of mammalian factor V. A mutant factor V that incorporated these five mutations (L2116M, S2117T, S2118T, E2119S, and S2183Y) (factor VMTTS/Y) was expressed in COS cells. After purification utilizing ion exchange chromatography, factor VMTTS/Y showed phospholipid-independent activity that could be inhibited with phospholipase A2. Subsequently, factor VMTTS/Y was washed extensively with CHAPS during purification to prevent phospholipid from co-purifying. Activity was measured with a prothrombin time assay with plasma lacking factor V. Specific activity was 1183 units/mg vs. 676 units/mg for wild type human factor V. Steady state kinetics of the prothrombinase complex with factor VMTTS/Y were assessed with varying concentrations of phospholipid vesicles. In the presence of membranes containing excess phosphatidylserine (15:20:65 PS:PE:PC), factor VMTTS/Y (5 pM) showed 39% greater Vmax than wild type human factor V and 3-fold higher apparent membrane affinity. With limiting phosphatidylserine (2:20:78 PS:PE:PC), factor VMTTS/Y (10 pM) showed 64% greater Vmax and 2-fold higher apparent membrane affinity. Factor VMTTS/Y, purified with a CHAPS wash, did not show lipid-independent activity but did support prothrombinase activity on membranes lacking PS or other negatively charged lipid (20:80 PE:PC). On these vesicles factor VMTTS/Y (50 pM) had a Vmax that was 8-fold higher than wild type factor V (see figure). These data indicate that the apparent phospholipid-independent activity results from higher membrane affinity or from greater activity on minimal phospholipid retained by factor V during purification. They imply that toxicity of pseutarin C may result, in part, from procoagulant activity on cell membranes that do not support the mammalian prothrombinase complex. Furthermore, they indicate that the precise manner in which the C2 domain of factor V binds to a phospholipid membrane influences the Vmax of the prothrombinase complex. Disclosures: No relevant conflicts of interest to declare.

Factor V Binding to Multimerin 1: Modulation by Factor V Activation and Binding Sites in the Factor V C1 and C2 Domains.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 193-193
Author(s):  
Samira B. Jeimy ◽  
Mary Ann Quinn-Allen ◽  
Nola Fuller ◽  
Kenneth Segers ◽  
Alan R. Stafford ◽  
...  
Keyword(s):  
Binding Sites ◽  
Thrombin Generation ◽  
Factor V ◽  
C2 Domain ◽  
Binding Studies ◽  
C2 Domains ◽  
Phospholipid Binding ◽  
Factor Va ◽  
Plasma Factor ◽  
C1 Domain

Abstract Platelets and endothelial cells store the polymeric factor V(a) binding protein, multimerin 1 (MMRN1), for release upon agonist stimulation. In human megakaryocytes, factor V binding to MMRN1 follows plasma factor V endocytosis, resulting in stored complexes of MMRN1 and factor V in platelet α-granules. The C2 domain of the factor V light chain contains a MMRN1 binding site; however, the affinity and stoichiometry of factor V-MMRN1 binding have not been determined, direct comparisons of factor V and Va binding to MMRN1 have not been done, and potential homologous roles of C1 and C2 domain structures in MMRN1 binding have not been studied. To further explore the mechanism of factor V and Va binding to MMRN1, and the roles of B domain release and C1 domain residues in MMRN1 binding, we used surface plasmon resonance and solid-phase binding studies. Functional consequences of factor V-MMRN1 binding were tested in competitive binding assays with the soluble phospholipid 1,2-Dicaproyl-sn-glycero-3-phospho-L-serine (C6PS), and calibrated automated thrombinography (CAT). Factor V bound to MMRN1 with a higher affinity than factor Va (approximately 2 nM versus 12 nM), and a stoichiometry consistent with binding to MMRN1 trimers. The higher affinity of factor V for MMRN1 was mainly due to differences in rates of formation of a more stable, secondary complex with MMRN1. Factor V activation by thrombin dissociated bound factor V from MMRN1, consistent with the reduced affinity of factor Va for MMRN1. A panel of point mutated, B domain deleted factor V constructs were used to identify MMRN1 binding residues in the C1 domain of factor V and Va. On a three dimensional model of factor Va, these residues mapped to a large, predominantly contiguous region between the C1 and C2 domains, that overlapped residues critical for factor Va phospholipid binding and procoagulant function. Consistent with the lowered affinity of factor Va for MMRN1, C6PS significantly inhibited factor Va-MMRN1, but not factor V-MMRN1 binding (p<0.05). Overlap between the MMRN1 and phospholipid binding sites was verified by CAT assays, as MMRN1 caused dose-dependent, significant reductions in plasma thrombin generation in these assays, by increasing lag time (p<0.01), and reducing peak (p<0.01) and total thrombin generation (p<0.01). Taken together, these data indicate that the functional homologies between the C domains of factor V extend to their MMRN1 binding sites. Moreover, thrombin has modulating effects on factor V-MMRN1 binding that mimic its effects on factor VIII-von Willebrand factor binding. The affinity of factor V-MMRN1 binding could be important to promote the association of MMRN1 with factor V in platelets, until factor V release and activation for prothrombinase assembly.

Thrombin-Catalyzed Cleavages of Factor VIII at Arg372 and Arg1689 Are Facilitated by the Acidic Residues Glu720-Asp725.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2686-2686
Author(s):  
Jennifer Newell ◽  
Qian Zhou ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Specific Activity ◽  
Factor Xa ◽  
Factor X ◽  
Wild Type ◽  
Factor Viiia ◽  
N Terminus ◽  
Acidic Residues ◽  
Type Factor ◽  
A2 Domain

Abstract Factor VIIIa acts as an essential cofactor for the serine protease factor IXa, together forming the Xase complex which catalyzes the conversion of factor X to factor Xa. The procofactor, factor VIII circulates as a heterodimeric protein comprised of a heavy chain (A1–A2-B domains) and a light chain (A3-C1-C2 domains) and is activated by proteolytic cleavage by thrombin at Arg372 (A1–A2 junction), Arg740 (A2-B junction), and Arg1689 (near the N-terminus of A3). The regions adjacent to the A1, A2, and A3 domains contain high concentrations of acidic residues and are designated a1 (residues 337–372), a2 (residues 711–740), and a3 (residues 1649–1689). In addition, the N-terminus of the A2 domain (residues 373–395) is rich in acidic residues, and results from a previous study revealed that this region contributes to the rate of thrombin-catalyzed cleavage at Arg740 (Nogami et. al., J. Biol. Chem. 280:18476, 2005). In this study we reveal a role for the acidic region following the A2 domain (a2, residues 717–725) in thrombin-catalyzed cleavage at both Arg372 and Arg1689. The factor VIII mutations Asp717Ala, Glu720Ala, Asp721Ala, Glu724Ala, Asp725Ala, and the double mutations of Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala were constructed, expressed, and purified from stably-transfected BHK cells as B-domainless protein. Specific activity values for the variants, relative to the wild type value were reduced to 70% for Asp717Ala; ∼50% for Glu720Ala, Asp721Ala, Glu724Ala, and Asp725Ala; and ∼30% for Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala. SDS-PAGE and western blotting of reactions containing the factor VIII variants and thrombin showed reductions in the rates of thrombin cleavage at both Arg372 and Arg1689 as compared to wild-type factor VIII. The cleavage rates for the single mutations comprising acidic residues 720–724 of factor VIII were reduced from ∼3-5-fold at Arg372, whereas this rate for the Asp717Ala mutant was similar to the wild-type value. The double mutations of Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala showed rate reductions of ∼7- and ∼27-fold, respectively at Arg372. While the rate for thrombin-catalyzed cleavage at Arg1689 in the Glu720Ala variant was similar to wild-type, rates for cleavage at this site were reduced ∼30-fold compared to wild-type factor VIII for the Asp721Ala, Glu724Ala, Asp725Ala, and Glu720Ala/Asp721Ala mutants, and ∼50-fold for the Glu724Ala/Asp725Ala variant. Furthermore, the generation of factor VIIIa activity following reaction with thrombin as assayed by factor Xa generation showed that all the mutants possessed peak activity values that were ∼2-3-fold reduced compared to wild type factor VIIIa. Moreover, in all the mutants the characteristic peak of activation was replaced with a slower forming, broad plateau of activity, with the double mutants showing the broadest activation profiles. These results suggest that residues Glu720, Asp721, Glu724, and Asp725 following the A2 domain modulate thrombin interactions with factor VIII facilitating cleavage at Arg372 and Arg1689 during procofactor activation.

Secretable Human Platelet-Derived Factor V Originates From the Plasma Pool

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3035-3041 ◽  
Author(s):  
Rodney M. Camire ◽  
Eleanor S. Pollak ◽  
Kenneth Kaushansky ◽  
Paula B. Tracy
Keyword(s):  
Western Blotting ◽  
Platelet Rich Plasma ◽  
Activated Protein C ◽  
Allogeneic Transplantation ◽  
Factor V ◽  
Wild Type ◽  
Prothrombinase Complex ◽  
Specific Association ◽  
American Society ◽  
Genetic Pools

Factor Va (FVa), derived from plasma or released from stimulated platelets, is the essential protein cofactor of the prothrombinase complex. Plasma-derived factor V (FV) is synthesized by the liver, whereas the source of the platelet-derived cofactor has not been unambiguously identified. Megakaryocytes, platelet precursors, are known to synthesize platelet proteins and to endocytose proteins from plasma (ie, fibrinogen) and then package these proteins into -granules. To determine which mechanism accounts for FV presence in platelets, two patients heterozygous for FVLeiden who underwent allogeneic transplantation from homozygous FV wild-type donors (bone marrow [BM] or liver) were studied. Patient JMW, whose skin biopsy specimen showed heterozygous FVLeiden, received a BM transplant from a wild-type homozygous FV donor as analyzed from posttransplant peripheral blood cells. Patient FW, whose native liver is heterozygous for FVLeiden, received a homozygous wild-type FV liver. Because each individual has two distinct genetic pools of factor V in liver and megakaryocytes, it was possible to determine whether secretable platelet-derived FV was normal or contained the FVLeiden mutation. Platelet-derived FVa released from thrombin-activated platelets from a normal individual, an individual heterozygous for the FVLeiden mutation, and the two patients was incubated with phospholipid vesicles and activated protein C (APC). Western blotting analyses using a monoclonal antibody that allows distinction between platelet-derived FVa and FVaLeiden subsequent to APC-catalyzed cleavage were then performed. Based on the accumulation of proteolytic fragments derived from APC-induced cleavage, analyses of platelet-derived FVa from JMW demonstrated both normal FVa and FVaLeiden consistent with a plasma-derived origin of the secretable platelet-derived FVa. Western blotting analyses of the APC-cleaved platelet-derived FVa from FW showed a wild-type phenotype, despite the presence of a FVLeiden allele in her megakaryocyte genome, also consistent with a plasma origin of her secretable platelet-derived FVa. Platelets do not appear to endocytose the plasma cofactor, because a 35-hour incubation of platelet-rich plasma with 125I-factor V showed no specific association/uptake of the radiolabeled ligand with the platelet pellet. Collectively, these results show for the first time that the majority of secretable platelet-derived factor V is endocytosed by megakaryocytes from plasma and is not exclusively synthesized by these cells, as previously believed. © 1998 by The American Society of Hematology.

Identification of the Multimerin Binding Region within the C2 Domain of Human Factor V Using Constructs Generated by Alanine-Scanning and Site-Directed Mutagenesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 124-124
Author(s):  
Samira B. Jeimy ◽  
Rachael A. Woram ◽  
Nola Fuller ◽  
Mary Anne Quinn-Allen ◽  
Gerard Nicolaes ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Light Chain ◽  
Coagulation Factor ◽  
Procoagulant Activity ◽  
Site Directed Mutagenesis ◽  
Factor V ◽  
C2 Domain ◽  
Wild Type ◽  
Factor Va ◽  
Covalently Linked

Abstract Activated coagulation factor V is a key non-enzymatic cofactor that is an essential component of the prothrombinase complex. In blood, much of the procoagulant factor V is stored in platelets, as a complex with the α-granule protein multimerin, for activation-induced release during clot formation. Presently, the molecular nature of multimerin - factor V binding has not been determined, although multimerin is known to interact with the light chain of factor V and Va. Using modified enzyme-linked immunoassays and recombinant factor V constructs, we previously found that discontinuous regions in the C2 domain of factor V were important for binding multimerin, and that these regions overlapped with areas in factor V important for its procoagulant function. Specifically, four (S2183T, W2063A/W2064A, K2060Q/K2061Q, K2060Q/K2061Q/W2063A/ W2064A) full-length, site-directed C2 mutants, and 12 (W2063A, W2064A (W2063, W2064)A, R2074A (R2072, R2074)A (K2101, K2103, K2104)A, L2116A (K2157, H2159, K2161)A, R2171A, R2174A, E2189A (R2187, E2189)A) B domain deleted, charge to alanine constructs had significantly reduced multimerin binding (p&lt; 0.01), relative to the corresponding wild-type. In the present study, we evaluated multimerin-factor V binding with a new assay that used affinity purified, recombinant multimerin immobilized onto microtitre wells to test the binding of recombinant factor V constructs. Because results from the new binding assays were in agreement on the regions of the C2 domain important for multimerin binding, the new assay was used to examine the effect of thrombin on factor V-multimerin binding. Thrombin exposure led to significant dissociation of preformed multimerin-factor V complexes (p&lt;0.01). In addition, thrombin cleaved factor Va had significantly reduced multimerin-binding in assays using antibodies against the factor Va heavy chain and light chain (p&lt;0.01). Recently, our lab identified that platelets contain forms of factor V covalently linked to multimerin via cysteine 1085 in the factor V B-domain. After recombinant factor V was activated by thrombin, there was no detectable binding of the liberated B-domain to multimerin (p&lt;0.001). Nonetheless, the B domain of factor V appeared to enhance factor V binding to multimerin, as factor V constructs synthesized without the B-domain had reduced multimerin binding even after conversion to factor Va, compared to wild-type factor V. Based on the overlap between multimerin-binding and procoagulant, PS binding regions in the C2 domain of factor V, we assessed the effect of multimerin on factor V procoagulant activity in one stage and two stage prothrombinase assays. However, multimerin did not neutralize factor V procoagulant activity when tested in molar excess. Our study indicates that multimerin binding of factor V is modulated by conformational changes in factor V upon activation, and that the factor V B-domain may function to enhance binding to multimerin. The dissociation of multimerin-factor V complexes by thrombin suggests multimerin might be important for delivering and localizing factor V onto platelets, prior to prothrombinase assembly.

Residues Surrounding Arg372, Arg740, and Arg1689 Contribute to the Rates of Thrombin-Catalyzed Cleavage of Factor VIII.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 847-847
Author(s):  
Jennifer L. Newell ◽  
Amy E. Griffiths ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Cleavage Site ◽  
Conflicts Of Interest ◽  
Specific Activity ◽  
Factor Xa ◽  
Wild Type ◽  
Cleavage Rate ◽  
Thrombin Cleavage ◽  
Type Factor ◽  
Bold Type

Abstract Abstract 847 Hemophilia A results from defects or deficiencies in the blood coagulation protein, factor VIII. Factor VIII circulates as an inactive procofactor that must be cleaved by thrombin or factor Xa at Arg740 (A2-B junction), Arg372 (A1-A2 junction), and Arg1689 (a3-A3 junction) to yield the active cofactor, factor VIIIa. Activation of factor VIII by thrombin is exosite-dependent yielding rates of cleavage at Arg740 ∼20-fold faster than Arg372, while cleavage at Arg1689 appears intermediary to Arg740 and Arg372. The contribution of P3-P3' residues flanking each cleavage site to the mechanism of thrombin-catalyzed cleavage of factor VIII has not been extensively studied. The P3-P3' residues for the 372, 1689, and 740 factor VIII sites are 370QIR*↓SVA375, 1687SPR*↓SFQ1692, and 738EPR*↓SFS743, respectively. Residues flanking Arg372 are considered non-optimal for thrombin cleavage with only two residues optimal (in bold type) for cleavage in the P3-P3' sequence, while residues flanking at the two other P1 sites are considered near-optimal with four out of six residues optimal (in bold type). Therefore, we investigated whether the P3-P3'residues surrounding Arg740, Arg372, and Arg1689 affect activation of factor VIII by thrombin. We constructed, stably transfected, and expressed four recombinant P3-P3' factor VIII mutants designated 372(P3-P3')740, 372(P3-P3')1689, 372(P3-P3')740/740(P3-P3')372, and 372(P3-P3')740/1689(P3-P3')372. For example, the 372(P3-P3')740 variant has replaced the non-optimal P3-P3' residues flanking Arg372 with the near-optimal P3-P3' residues flanking Arg740. The specific activities of the 372(P3-P3')740 and 372(P3-P3')740/740(P3-P3')372 mutants were 98% and 122% the wild-type factor VIII value, respectively. In comparison, the 372(P3-P3')1689 and 372(P3-P3')740/1689(P3-P3')372 showed reductions in specific activity with values that were 14% and 17% of wild-type factor VIII, consistent with possible impaired rates of activation by thrombin. SDS-PAGE and Western blotting of the three variants possessing the 372(P3-P3')740 mutation showed cleavage rates at Arg372 increased 11- to 14-fold compared with wild-type factor VIII as judged by rates of generation of the A1 subunit. Furthermore, these variants revealed 11-21-fold rate increases in the generation of the A2 subunit as compared to wild-type factor VIII. The rates of A1 and A2 subunit generation were moderately increased from 2-3-fold for the 372(P3-P3')1689 mutant. These results indicate that replacing the non-optimal residues flanking Arg372 with near-optimal residues enhances rates of cleavage at this site. Furthermore, since the P2-P2' residues flanking Arg740 and Arg1689 are identical, these results also suggest that the P3 and/or P3' residues from the Arg740 cleavage site make a greater contribution to the enhanced cleavage rate when inserted at Arg372 than the equivalent residues from the Arg1689 site. Thrombin cleavage of light chain showing the largest effect was obtained for the 372(P3-P3')740/1689(P3-P3')372 mutant which yielded a reduced rate of A3-C1-C2 subunit generation by 33-fold. This result suggests that replacing near-optimal P3-P3' residues at Arg1689 with non-optimal residues at Arg372 significantly reduces the rate of thrombin cleavage at Arg1689, an effect that may contribute to its low specific activity. There was no observed defect in Arg1689 cleavage in the 372(P3-P3')740 mutant and moderate 2-3-fold reductions in thrombin-catalyzed cleavage rates at Arg1689 in the 372(P3-P3')1689, 372(P3-P3')740/740(P3-P3')372, and 372(P3-P3')740 variants. Overall, these results suggest that faster cleavage rates at Arg740 and Arg1689 can be attributed to more optimal residues in the P3-P3' region, while the relatively slower cleavage rate at Arg372 can be accelerated by replacement with more optimal residues for thrombin cleavage. Thus, the P3-P3' residues surrounding Arg740, Arg1689, and Arg372 in factor VIII impact rates of thrombin proteolysis at each site and contribute to the mechanism for thrombin activation of the procofactor. Disclosures: No relevant conflicts of interest to declare.

Gla domain–mutated human protein C exhibiting enhanced anticoagulant activity and increased phospholipid binding

Blood ◽  
2003 ◽  
Vol 101 (6) ◽  
pp. 2277-2284 ◽  
Author(s):  
Yong-Hui Sun ◽  
Lei Shen ◽  
Björn Dahlbäck
Keyword(s):  
Protein C ◽  
Molecular Mechanisms ◽  
Therapeutic Potential ◽  
Anticoagulant Activity ◽  
Protein S ◽  
Phospholipid Membranes ◽  
Factor V ◽  
Phospholipid Binding ◽  
Membrane Affinity ◽  
Gla Domain

Protein C is a member of the vitamin K– dependent protein family. Proteins in this family have similar γ-carboxyglutamic acid (Gla)–rich domains, but their affinities for negatively charged phospholipid membranes vary more than 1000-fold. We have shown that it is possible to enhance anticoagulant activity and membrane affinity of protein C by selective mutagenesis of the Gla domain. In this study, 3 new mutants, Q10G11N12 (QGN), S23E32D33Y44 (SEDY), and Q10G11N12S23E32D33Y44 (QGNSEDY), were created. In plasma-based coagulation assays, the activated form of QGNSEDY (QGNSEDY-APC) demonstrated approximately 20-fold higher anticoagulant activity than wild-type activated protein C (WT APC), while QGN-APC and SEDY-APC did not. Both normal activated factor V (FVa) and FVa Leiden (Arg506Gln) were degraded much more efficiently by QGNSEDY-APC than by WT APC in the presence as well as in the absence of protein S. Binding of protein C variants to negatively charged phospholipid membranes was investigated using light scattering and the BIAcore technique. QGNSEDY demonstrated 3- to 7-fold enhanced binding as compared with WT protein C, suggesting the membrane affinity to be influenced by several residues located at different parts of the Gla domain. The anticoagulant activity as well as phospholipid binding ability was only enhanced when multiple regions of the Gla domain were modified. The results provide insights into the molecular mechanisms that are involved in determining the binding affinity of the interaction between Gla domains and phospholipid membranes. The unique properties of QGNSEDY-APC suggest this APC variant possibly to have greater therapeutic potential than WT APC.

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