PubMed Health⌕ Search

Biomedical subjects

M Kalafatis

Publications and source records attributed to M Kalafatis.

At least 19 recordsLinked to original sources
PubMed1997

An in vitro analysis of the combination of hemophilia A and factor V(LEIDEN).

The classification of factor VIII deficiency, generally used based on plasma levels of factor VIII, consists of severe (<1% normal factor VIII activity), moderate (1% to 4% factor VIII activity), or mild (5% to 25% factor VIII activity). A recent communication described four individuals bearing identical factor VIII mutations. This resulted in a severe bleeding disorder in two patients who carried a normal factor V gene, whereas the two patients who did not display severe hemophilia were heterozygous for the factor V(LEIDEN) mutation, which leads to the substitution of Arg506 --> Gln mutation in the factor V molecule. Based on the factor VIII level measured using factor VIII-deficient plasma, these two patients were classified as mild/moderate hemophiliacs. We studied the condition of moderate to severe hemophilia A combined with the factor V(LEIDEN) mutation in vitro in a reconstituted model of the tissue factor pathway to thrombin. In the model, thrombin generation was initiated by relipidated tissue factor and factor VIIa in the presence of the coagulation factors X, IX, II, V, and VIII and the inhibitors tissue factor pathway inhibitor, antithrombin-III, and protein C. At 5 pmol/L initiating factor VIIa x tissue factor, a 10-fold higher peak level of thrombin formation (350 nmol/L), was observed in the system in the presence of plasma levels of factor VIII compared with reactions without factor VIII. Significant increase in thrombin formation was observed at factor VIII concentrations less than 42 pmol/L (approximately 6% of the normal factor VIII plasma concentration). In reactions without factor VIII, in which thrombin generation was downregulated by the addition of protein C and thrombomodulin, an increase of thrombin formation was observed with the factor V(LEIDEN) mutation. The level of increase in thrombin generation in the hemophilia A situation was found to be dependent on the factor V(LEIDEN) concentration. When the factor V(LEIDEN) concentration was varied from 50% to 150% of the normal plasma concentration, the increase in thrombin generation ranged from threefold to sevenfold. The data suggested that the analysis of the factor V genotype should be accompanied by a quantitative analysis of the plasma factor V(LEIDEN) level to understand the effect of factor V(LEIDEN) in hemophilia A patients. The presented data support the hypothesis that the factor V(LEIDEN) mutation can increase thrombin formation in severe hemophilia A.

Factor V↗
PubMed1997

Activated protein C cleavage of factor Va leads to dissociation of the A2 domain.

The products of cleavage of bovine factor Va by activated protein C (APC) in the presence and absence of phospholipid (25% phosphatidylserine, 75% phosphatidylcholine, PCPS) were evaluated using sedimentation velocity/equilibrium methods in the analytical ultracentrifuge and by immunoprecipitation using an antibody directed against the light chain of the factor Va molecule. The molecular weight and sedimentation coefficient of the associated heavy and light chains of factor Va, 173,000 (7.9 S) is reduced to 132,000 (7.1 S) by APC cleavage at Arg505 and Arg662. Complete cleavage of the factor Va heavy chain (with APC-PCPS) at Arg505, Arg662 and Arg306 results in a drastic change in the molecular weight observed for the product. Two products are resolved with sedimentation coefficients of 3.3 and 6.3 S with estimated molecular weights of 48,000 and 114,000, respectively. Immunoprecipitation studies showed that the products of factor Va cleavage at Arg505 and Arg662 (A1A2N.A2C.LC) are mostly noncovalently associated and consequently immunoprecipitated with an antibody directed against the light chain of the factor Va molecule. In contrast, for factor Va cleaved at Arg505, Arg662, and Arg306 the precipitated complex consisted of the A1 domain (residues 1-306) and the light chain (residues 1537-2183) of factor Va (A1.LC). The fragments corresponding to residues 307-505 (A2N) and 506-662 (A2C) are found in the supernatant. The combined mass of these two products (48,000) is similar to the estimated mass of the 3.3 S fragment estimated from sedimentation velocity/equilibrium studies; while the combined mass of the 1-306 + 1537-2183 products corresponds to 114,000, the estimated mass of the 6.3 S fragment. These data lead to the conclusion that cleavages at Arg306, Arg505, and Arg662 of the factor Va molecule resulted in the dissociation of the entire A2 domain as two noncovalently associated fragments (A2N.A2C). Enzyme kinetic and light scattering data suggest that the complete inactivation of the factor Va molecule involves not only cleavage at Arg306 but also the dissociation of the A2 domain. These data also suggest that the complete APC inactivation of the factor Va molecule is analogous to the spontaneous inactivation of factor VIIIa, which occurs via the dissociation of the A2 domain.

Animals↗
PubMed1997

Increased tissue factor-initiated prothrombin activation as a result of the Arg506 --> Gln mutation in factor VLEIDEN.

The effect of the Arg506 --> Gln mutation in factor VLEIDEN on thrombin generation was evaluated in a reconstituted system using the purified components of the tissue factor (TF) pathway to thrombin and the components of the protein C pathway. Recombinant full-length tissue factor pathway inhibitor (RTFPI) was included in the system because of a previously observed synergistic inhibitory effect of TFPI and the protein C pathway on TF-initiated thrombin generation. Thrombin generation initiated by 1.25 pM factor VIIa.TF in the absence of the protein C pathway components occurs following an initiation phase, after which prothrombin is quantitatively converted to 1.4 microM thrombin. The factor VLEIDEN mutation did not influence thrombin generation in the reconstituted model in the absence of the protein C pathway. In the presence of 2.5 nM TFPI, 65 nM protein C, and 10 nM recombinant soluble thrombomodulin (Tm), thrombin generation catalyzed by normal factor V was abolished after the initial formation of 25 nM thrombin. In contrast, persistent thrombin generation was observed in the presence of factor VLEIDEN in the same system, although the rate of thrombin generation was slower compared with the reaction without protein C and Tm. The rate of thrombin generation with factor VLEIDEN increased with time and ultimately resulted in quantitative prothrombin activation. When the TFPI concentration was reduced to 1.25 nM, thrombin generation is still curtailed in the presence of normal factor V. In contrast, under similar conditions using factor VLEIDEN, the protein C pathway totally failed to down-regulate thrombin generation. The dramatic effect of a 50% reduction in TFPI concentration on the inhibitory potential of the protein C pathway on thrombin generation catalyzed by factor VLEIDEN suggests that the observed synergy between TFPI and the protein C pathway is directly governed by the TFPI concentration and by cleavage of the factor Va heavy chain at Arg506. This cleavage appears to have a dramatic regulatory effect in the presence of low concentrations of TFPI. Markedly increased thrombin generation in the presence of both 1.25 nM TFPI and factor VLEIDEN was also observed when antithrombin-III was added to the system to complete the natural set of coagulation inhibitors. Protein S (300 nM) had a minimal effect in the model on the inhibition of thrombin generation by protein C, Tm, and TFPI, with either normal factor V or factor VLEIDEN. Protein S also failed to significantly potentiate the action of the protein C pathway in the presence of antithrombin-III in reactions employing normal factor V or factor VLEIDEN. The absence of an effect of protein S in the model, which employs saturating concentrations of phospholipid, suggests that the reported interactions of protein S with coagulation factors are not decisive in the reaction. Altogether the data predict that TFPI levels in the lower range of normal values are a risk factor for thrombosis when combined with the Arg506 --> Gln mutation in factor VLEIDEN.

Animals↗
PubMed1997

Inhibitory mechanism of the protein C pathway on tissue factor-induced thrombin generation. Synergistic effect in combination with tissue factor pathway inhibitor.

The effects of the components of the protein C pathway on thrombin generation were studied in a reconstituted model in which thrombin is generated by factor VIIa and relipidated tissue factor (TF) via the activation of the purified coagulation factors X, IX, VIII, V, and prothrombin. The influence of protein C and soluble thrombomodulin on thrombin generation was correlated with factor Xa generation, factor V(a) and factor VIII(a) formation/inactivation, and protein C activation. Thrombin generation initiated by low concentrations of factor VIIa.TF (1.25 pM) occurs in an explosive fashion during a propagation phase which occurs after an initiation phase of approximately 1 min in which only traces of thrombin are formed. In the absence of other inhibitors, protein C (65 nM) in combination with high concentrations of soluble thrombomodulin (10 nM) resulted in a reduced rate of thrombin generation during the propagation phase without affecting the initiation phase; the activated protein C generated failed to neutralize prothrombinase activity and did not prevent prothrombin consumption. In the presence of plasma levels of the tissue factor pathway inhibitor (2. 5 nM recombinant TFPI), the protein C pathway reduced the rate of thrombin generation, initiated by 1.25 pM factor VIIa.TF, and completely eliminated prothrombinase activity at soluble thrombomodulin concentrations of >/=1 nM. The neutralization of prothrombinase activity coincided with cleavages at Arg-506 and subsequent cleavage at Arg-306 of the factor Va heavy chain by activated protein C. Thus, the protein C pathway combined with TFPI creates a minimal inhibitory potential required to shut down TF-initiated thrombin generation. The protein C pathway constituents did not influence factor Xa generation or factor VIIIa degradation over the interval in which prothrombinase activity was neutralized. Our data thus suggest that the protein C pathway regulates thrombin generation solely by the inactivation of factor Va. At low initiating factor VIIa.TF (1.25 pM) and high thrombomodulin concentrations (10 nM), the factor Va heavy chain is cleaved before significant amounts of light chain are generated. The ability of the protein C pathway to inhibit thrombin generation was greatly reduced when the reaction was initiated in the presence of factor Va, supporting the hypothesis that effective down-regulation of thrombin generation by the protein C pathway, in reactions initiated with the procofactor, occurs by prevention of the coexistence of the factor Va heavy and light chains.

Drug Synergism↗
PubMed1997

Analysis of tissue plasminogen activator specificity using peptidyl fluorogenic substrates.

A series of 54 fluorogenic substrates have been synthesized and evaluated for tissue-type plasminogen activator (tPA) hydrolysis in an attempt to create efficient sensitive substrates for tPA and to investigate substrate structure-efficiency correlations. All substrates contain the 6-amino-1-naphthalenesulfonamide (ANSN) leaving group, Arg in the P1 position, various amino acids in the P2 and P3 positions, and various substituents in the sulfonamide moiety of the leaving group (P' position). The majority of substrates have relatively low K(M) values (< 100 microM), reaching as low as 2.6 microM, and reasonably high k(cat) values (up to 3.6 s(-1)). These substrates have higher affinity, higher hydrolysis rates, and higher efficiency for two-chain tPA than for the single-chain form of this enzyme. Analysis of the P3 structure influence on substrate efficiency demonstrates that compounds which contain D-isomers of N-blocked bulky amino acids, such as Phe, Leu, and Val, in this position are more efficient for tPA than substrates with N-unblocked small amino acids (Ser or Pro) in the P3 position. The second-order rate constants and k(cat) values for substrate hydrolysis increase with decreases in the P2 amino acid hydrophobicity in the following manner: Leu < Val and Gly < Ser < Pro. Substrates which contain an ANSN leaving group had a higher affinity for tPA than substrates with p-nitroaniline or 7-amino-4-methylcoumarin leaving groups. Analyses of substrate hydrolysis dependence on the substrate P' structure show that the k(cat) and the second-order rate constants increased with an increase in the size of monoalkyl substituent in the sulfonamide moiety, whereas substrates which contain either glycine methyl ester or a dialkyl group displayed the lowest efficiency for tPA. The substrate Boc-(p-F)Phe-Pro-Arg-ANSNHC2H5 allowed quantitation of tPA at a concentration as low as 1 pM, a concentration significantly lower than the plasma concentration of this protein. Evaluation of the activation of single-chain tPA by factor Xa demonstrates that prothrombinase is approximately 3-fold more efficient in activating sc-tPA than factor Xa alone, increasing the initial rate of activation from 0.0055 nM/s per 1 nM of factor Xa to 0.017 nM/s per 1 nM.

Animals↗
PubMed1997

The effect of Arg306-->Ala and Arg506-->Gln substitutions in the inactivation of recombinant human factor Va by activated protein C and protein S.

Factor Va (fVa) is inactivated by activated protein C (APC) by cleavage of the heavy chain at Arg306, Arg506, and Arg679. Site-directed mutagenesis of human factor V cDNA was used to substitute Arg306-->Ala (rfVa306A) and Arg506-->Gln (rfVa506Q). Both the single and double mutants (rfVa306A/506Q) were constructed. The activation of these procofactors by alpha-thrombin and their inactivation by APC were assessed in coagulation assays using factor V-deficient plasma. All recombinant and wild-type proteins had similar initial cofactor activity and identical activation products (a factor Va molecule composed of light and heavy chains). Inactivation of factor Va purified from human plasma (fVaPLASMA) in HBS Ca2+ +0.5% BSA or in conditioned media by APC in the presence of phospholipid vesicles resulted in identical inactivation profiles and displayed identical cleavage patterns. Recombinant wild-type factor Va (rfVaWT) was inactivated by APC in the presence of phospholipid vesicles at an overall rate slower than fVaPLASMA. The rfVa306A and rfVa506Q mutants were each inactivated at rates slower than rfVaWT and fVaPLASMA. Following a 90-min incubation with APC, rfVa306A and rfVa506Q retain approximately 30-40% of the initial cofactor activity. The double mutant, rfVa306A/506Q, was completely resistant to cleavage and inactivation by APC retaining 100% of the initial cofactor activity following a 90-min incubation in the presence of APC. Recombinant fVaWT, rfVa306A, rfVa506Q, and rfVa306A/506Q were also used to evaluate the effect of protein S on the individual cleavage sites of the cofactor by APC. The initial rates of rfVaWT and rfVa306A inactivation in the presence of protein S were unchanged, indicating cleavage at Arg506 is not affected by protein S. The initial rate of rfVa506Q inactivation was increased, suggesting protein S slightly accelerates the cleavage at Arg306. Overall, the data demonstrate high specificity with respect to cleavage sites for APC on factor Va and demonstrate that cleavages of the cofactor at both Arg306 and Arg506 are required for efficient factor Va inactivation.

Alanine↗
PubMed1997

Protein C activation and factor Va inactivation on human umbilical vein endothelial cells.

The inactivation of factor Va was examined on primary cultures of human umbilical vein endothelial cells (HUVECs), either after addition of activated protein C (APC) or after addition of alpha-thrombin and protein C (PC) zymogen. Factor Va proteolysis was visualized by Western blot analysis using a monoclonal antibody (alpha HVaHC No. 17) to the factor Va heavy chain (HC), and cofactor activity was followed both in a clotting assay using factor V-deficient plasma and by quantitation of prothrombinase function. APC generation was monitored using the substrate 6-(D-VPR)amino-1-naphthalenebutylsulfonamide (D-VPR-ANSNHC4H9), which permits quantitation of APC at 10 pmol/L. Addition of APC (5 nmol/L) to an adherent HUVEC monolayer (3.5 x 10(5) cells per well) resulted in a 75% inactivation of factor Va (20 nmol/L) within 10 minutes, with complete loss of cofactor activity within 2 hours. Measurements of the rate of cleavage at Arg506 and Arg306 in the presence and absence of the HUVEC monolayer indicated that the APC-dependent cleavage of the factor Va HC at Arg506 was accelerated in the presence of HUVECs, while cleavage at Arg306 was dependent on the presence of the HUVEC surface. Factor Va inactivation proceeded with initial cleavage of the factor Va HC at Arg506, generating an M(r) 75,000 species. Further proteolysis at Arg306 generated an M(r) 30,000 product. When protein C (0.5 mumol/L), alpha-thrombin (1 nmol/L), and factor Va (20 nmol/L) were added to HUVECs an APC generation rate of 1.56 +/- 0.11 x 10(-14) mol/min per cell was observed. With APC generated in situ, cleavage at Arg506 on the HUVEC surface is followed by cleavage at Arg306, generating M(r) 75,000 and M(r) 30,000 fragments, respectively. In addition, the appearance of two novel products derived from the factor Va HC are observed when thrombin is present on the HUVEC surface: the HC is processed through limited thrombin proteolysis to generate an M(r) 97,000 fragment, which is further processed by APC to generate an M(r) 43,000 fragment. NH2-terminal sequence analysis of the M(r) 97,000 fragment revealed that the thrombin cleavage occurs in the COOH-terminus of the intact factor Va HC since both the intact HC as well as the M(r) 97,000 fragment have the same sequence. Our data demonstrate that the inactivation of factor Va on the HUVEC surface, initiated either by APC addition or PC activation, follows a mechanism whereby cleavage is observed first at Arg506 followed by a second cleavage at Arg306. The latter cleavage is dependent on the availability of the HUVEC surface. This mechanism of inactivation of factor Va is similar to that observed on synthetic phospholipid vesicles.

Blotting, Western↗
PubMed1997

The regulation of clotting factors.

Blood clotting involves a multitude of proteins that act in concert in response to vascular injury to produce the procoagulant enzyme alpha-thrombin, which in turn is responsible for the generation of the fibrin plug. However, while generation of the fibrin plug is required for the arrest of excessive bleeding, unregulated clotting will result in the occlusion of the blood vessels and thrombosis. Thus, the regulation of the delicate balance between the procoagulant and anticoagulant mechanisms is of extreme importance for survival. While the majority of proteins involved in blood coagulation circulate as inactive zymogens that require proteolytic activation in order to function, approximately 1% of the circulating factor VII molecules are active. Factor VIIa, possess a serine protease active site, has poor catalytic activity, and is not inhibited by the circulating stoichiometric protease inhibitors. Following injury to the vasculature and subsequent exposure of the integral membrane glycoprotein, tissue factor (TF), the circulating factor VIIa molecules can bind to the exposed TF forming the extrinsic tenase complex (TF/factor VIIa) and initiate the blood coagulation process. Formation of the TF/factor VIIa complex increases the catalytic efficiency of the enzyme by four orders of magnitude when compared with factor VIIa alone. This cell-associated enzymatic complex initiates a series of enzymatic reactions, leading to the generation of alpha-thrombin and ultimately to the formation of the fibrin plug. The procoagulant enzymatic complexes (i.e., prothrombinase, intrinsic tenase, and extrinsic tenase) are similar in structure and composed of an enzyme, a cofactor, and the substrate associated on a cell surface in the presence of divalent metal ions. While the activity of the extrinsic tenase complex is limited by the availability (exposure) of its cell-associated cofactor (TF) it is remarkable that the activities of both the prothrombinase complex (factor Va/factor Xa) as well as the intrinsic tenase complex (factor VIIIa/factor IXa) are limited by the presence of the two soluble, nonenzymatic cofactors, factor Va and factor VIIIa. Factor Va and factor VIIIa, which are very similar in structure and function, are required for prothrombinase and intrinsic tenase activities, respectively, because both cofactors express a dual function in their respective complexes, acting as an enzyme receptor and catalytic effector on the cell surface. The cofactors derive from inactive plasma precursors by regulatory proteolytic events that involve alpha-thrombin. In general, bleeding tendencies are usually associated with defects in the activation of one of the zymogens or the cofactors of the procoagulant complexes. However, the activity of all of the complexes is also limited by the availability of an adequate membrane surface provided by endothelial cells, platelets, and monocytes. The cell surface provides a site for the recruitment of the appropriate proteins and allows for fast and efficient clot formation. In the absence of an appropriate membrane surface, the procoagulant complexes have limited catalytic efficiency. Thus, timely exposure of the adequate membrane surface is an additional step in the regulation of alpha-thrombin formation. alpha-Thrombin participates in its own down-regulation by binding to the endothelial cell receptor thrombomodulin, initiating the protein C pathway, which in turn leads to the formation of activated protein C (APC). APC is required for efficient neutralization of factor Va cofactor activity, which results in the inactivation of the prothrombin-activating complex. This inactivation can only occur in the presence of the appropriate membrane surface. Thus, while following alpha-thrombin activation, factor VIIIa is rapidly and spontaneously inactivated by dissociation of the A2 domain from the rest of the cofactor, APC is required for down-regulation of alpha-thrombin formation by prothrombinase. (ABSTRACT

Blood Coagulation Factors↗
PubMed1996

An antifibrinolytic mechanism describing the prothrombotic effect associated with factor VLeiden.

Factor Va is the essential cofactor in prothrombinase-dependent activation of prothrombin. Resistance of Factor VaLeiden to inactivation by activated protein C (APC) contributes to thrombotic tendencies in subjects with the variant due, in part, to the inability to terminate thrombin production which increases both fibrin accretion and the frequency of thrombus formation. A reduced ability to inhibit thrombin generation, however, may lead to the stabilization of a clot through the activation of thrombin activatable fibrinolysis inhibitor (TAFI). This hypothesis was tested by determining the profibrinolytic effect of APC on lysis time using clots formed with plasma from either homozygous normal (n = 4) or homozygous factor VLeiden (n = 4) subjects. Clots were formed in the presence of tissue-type plasminogen activator, thrombin, phosphatidylcholine/phosphatidylserine vesicles, Ca2+, and various concentrations of APC. Approximately 10-fold more APC was required to reduce lysis time from 140 to 50 min in clots containing factor VLeiden compared to normal factor V. This effect was specific to the form of factor V present in plasma since identical results were obtained in an appropriately reconstituted purified system, which included both TAFI and either form of factor V purified from pooled plasma. In the absence of TAFI, APC did not affect clot lysis in experiments with either normal factor V or factor VLeiden. During the various lysis assays performed with purified components, clots were solubilized and the proteolytic alterations in factor V/Va were assessed by Western blotting using a specific factor Va heavy chain monoclonal antibody. The heavy chain of factor VaLeiden persisted for as long as 60 min, in the presence of 6.3 n APC indicating sustained activity of factor VaLeiden during the lysis assay. In contrast, no factor Va heavy chain was present after the first 5.0 min in clots formed in the presence of normal factor V and 6.3 n APC. These combined data indicate that factor VaLeiden specifically attenuates the profibrinolytic effect of APC. Thus, an impaired TAFI-dependent profibrinolytic response to APC in APC-resistant individuals appears to be an additional factor contributing to the prothrombotic tendencies in subjects with factor VLeiden.

Carboxypeptidase B2↗
PubMed1996

Compound heterozygous protein C deficiency resulting in the presence of only the beta-form of protein C in plasma.

About 30% of human plasma protein C (PC) is of lower molecular weight than the predominant alpha-form. The minor beta-form arises as a consequence of the lack of glycosylation at Asn329. Although the functional role of Asn329 has been investigated by in vitro mutagenesis, until now no naturally occurring mutations have been reported at this site. We describe here the case of two identical twin sisters compound heterozygous for two novel PC mutations: Cys78-->Stop inherited from the maternal side and Asn329-->Thr inherited from the paternal side, associated with the presence of only the beta-form of PC in plasma. The Cys78-->Stop substitution is predicted to abolish PC synthesis from one allele, whereas the Asn329-->Thr substitution results in the reduced synthesis of a beta PC variant with decreased functional activity. PCN329T from the two monovular twin sisters was purified and its active form APCN329T was assessed for its ability to inactivate factor Va. Whereas no differences were observed between the activation rates of normal PC and PCN329T, APCN329T inactivated human factor Va with a rate slower than the normal APC. This is the first report of a PC defect involving glycosylation of the molecule. This defect results in the presence of only the beta-form of PC in human plasma and is responsible for the reduced anticoagulant activity observed.

Adult↗
PubMed1996

Proteolytic events that regulate factor V activity in whole plasma from normal and activated protein C (APC)-resistant individuals during clotting: an insight into the APC-resistance assay.

Human factor V is activated to factor Va by alpha-thrombin after cleavages at Arg709, Arg1018, and Arg1545. Factor Va is inactivated by activated protein C (APC) in the presence of a membrane surface after three sequential cleavages of the heavy chain. Cleavage at Arg506 provides for efficient exposure of the inactivating cleavages at Arg306 and Arg679. Membrane-bound factor V is also inactivated by APC after cleavage at Arg306. Resistance to APC is associated with a single nucleotide change in the factor V gene (G1691-->A) corresponding to a single amino acid substitution in the factor V molecule: Arg506-->Gln (factor V Leiden). The consequence of this mutation is a delay in factor Va inactivation. Thus, the success of the APC-resistance assay is based on the fortuitous activation of factor V during the assay. Plasmas from normal individuals (1691 GG) and individuals homozygous for the factor V mutation (1691 AA) were diluted in a buffer containing 5 mmol/L CaCl2, phospholipid vesicles (10 micromol/L), and APC. APC, at concentrations < or = 5.5 nmol/L, prevented clot formation in normal plasma, whereas under similar conditions, a clot was observed in plasma from APC-resistant individuals. Gel electrophoresis analyses of factor V fragments showed that membrane-bound factor V is primarily cleaved at Arg306 in both plasmas. However, whereas in normal plasma production of factor Va heavy chain is counterbalanced by fast degradation after cleavage at Arg506/Arg306, in the APC-resistant individuals' plasma, early generation and accumulation of the heavy chain portion of factor Va occurs as a consequence of delayed cleavage at Arg306. At elevated APC concentrations (>5.5 nmol/L), no clot formation was observed in either plasma from normal or APC-resistant individuals. Our data show that resistance to APC in patients with the Arg506-->Gln mutation is due to the inefficient degradation (inactivation) of factor Va heavy chain by APC.

Amino Acid Sequence↗
PubMed1996

Comparison of activated protein C/protein S-mediated inactivation of human factor VIII and factor V.

The proteolytic cleavage and subsequent inactivation of recombinant human factor VIII (rhFVIII) and human factor VIIIa (rhFVIIIa) by recombinant human activated protein C (rAPC) was analyzed in the presence and absence of human protein S and human factor V (FV). Membrane-bound rhFVIIIa spontaneously looses most of its initial cofactor activity after 15 minutes of incubation at pH 7.4. The remaining activity can be eliminated after incubation with rAPC. Complete inactivation of the membrane-bound rhFVIII and rhFVIIIa by APC correlates with cleavage at Arg336. The inactivation of rhFVIII and human plasma FV by rAPC were also compared. Under similar experimental conditions, complete inactivation of membrane-bound FVIII (60 nmol/L) by rAPC (10 nmol/L) requires 4 hours of incubation, in contrast to 5 minutes for FV (60 nmol/L). The presence of protein S (100 nmol/L) enhances rhFVIII inactivation by rAPC by 6.4-fold and FVa inactivation by twofold, whereas membrane-bound FV showed no protein S dependence during inactivation. The addition of human FV to the APC/protein S inactivation mixture increases by approximately twofold the rate of inactivation of rhFVIII. The effect of FV on the rhFVIII inactivation by APC is protein S-dependent, because FV alone has no effect on the inactivation rate of rhFVIII by APC. Western blotting using a monoclonal antibody that recognizes an epitope between amino acid residues 307 and 506 of human FV showed that FV was completely cleaved by APC at the beginning of the rhFVIII inactivation process. These data suggest that FV fragments derived from the B region of the procofactor after incubation of the membrane-bound procofactor with APC, but not intact single-chain FV, stimulate APC activity in the presence of protein S. rhFVIII, FV, and rhFVIIIa were not inactivated by Glu20-->Ala-substituted rAPC (rAPCgamma20A), and membrane-bound factor Va was only partially inactivated. Our data suggest that (1) FV and FVa are the physiologically significant substrates for APC inactivation and (2) membranes-bound APC-treated FV is a cofactor for the APC inactivation of rhFVIII only in the presence of the intact form of protein S.

Factor VIIIa↗
PubMed1996

Regulation and regulatory role of gamma-carboxyglutamic acid containing clotting factors.

Blood coagulation is initiated following damage of the vessel wall. The large number of proteins that participate in the reactions that regulate blood coagulation must behave in a concerted manner to generate alpha-thrombin. This most abundant enzyme product of the coagulation process acts on the soluble blood protein fibrinogen to create the insoluble fibrin clot, trigger platelet activation, and initiate a variety of vascular processes ranging from coagulation inhibition and activation of fibrinolysis to cell growth. The majority of enzymes that participate in the blood-clotting process circulate in blood as inactive zymogens and procofactors. Following mechanical injury to the vessel wall, a cell-associated protein, tissue factor, is exposed and initiates a series of enzymatic reactions localized on a membrane surface generally provided by cells and accumulating platelets. The binding of the enzymes and zymogen substrates to the exposed membrane surface and their activation are in part governed by the gamma-carboxyglutamic acid residues (gla) of each protein. Recently, it has been demonstrated that the gla residues are necessary for Ca2+ binding and subsequent exposure of the hydrophobic membrane binding regions to the cell bilayer. Thus, the gla residues play a preeminent role during the blood-clotting process. Absence of gamma-carboxylation of one or more of the critical glutamic acid residues results in an impaired coagulation/anticoagulation process which may lead to a bleeding diathesis or thrombosis.

1-Carboxyglutamic Acid↗
PubMed1995

The mechanism of inactivation of human platelet factor Va from normal and activated protein C-resistant individuals.

The inactivation of human platelet factor Va by activated protein C (APC) was analyzed by functional assessment of cofactor activity and Western blotting analysis to visualize the factor Va fragments accompanying proteolysis. Platelets were treated with thrombin to facilitate both their activation as well as the release and further activation of platelet factor Va, followed by APC addition. The rates of inactivation were donor-dependent such that 15-60% of the initial cofactor activity was lost within 5 min of APC addition with as much as 10-20% of the activity still remaining after 2 h of incubation. Western blot analysis using a monoclonal antibody that recognizes an epitope between amino acid residues 307 and 506 of the factor V molecule suggested that the factor Va activity resistant to APC inactivation was due to residual heavy chain. Furthermore, in contrast to studies with normal plasma-derived factor Va, two possible cleavage mechanisms could explain the platelet factor Va fragments observed. APC can cleave platelet factor Va initially at Arg506, with subsequent cleavages occurring at Arg306 and Arg679. Alternatively, APC can cleave at Arg306 initially, with further cleavage at Arg679 then at Arg506 or at Arg506 followed by cleavage at Arg679. Similar results were obtained if platelets were removed from the inactivation mixtures and phospholipid vesicles were used to supply the membrane surface required for inactivation, suggesting that the order of platelet factor Va peptide bond cleavage or the amount of cofactor activity remaining was not altered by either of these surfaces. Thus, APC is unable to effect the complete inactivation of platelet factor Va, even though it would appear that the same cleavages which render the plasma cofactor inactive are occurring in the platelet cofactor. Analogous protocols were used to study an individual heterozygous for the Arg506-->Gln506 mutation (Factor V Leiden, Factor VR506Q). With respect to the mutant platelet factor Va in the presence of APC, > 70% of the initial cofactor activity remained after 1 min, with 30% activity still remaining after 2 h. As seen in studies of the APC-catalyzed inactivation of plasma factor VaR506Q, proteolysis of the mutant platelet factor Va confirms that even though cleavage at Arg306 will occur in the absence of cleavage at Arg506, the rate of inactivation is slower. Collectively these data suggest that when compared to normal plasma factor Va, differences in normal platelet factor Va which define: 1) whether the heavy chain is susceptible to cleavage at Arg306 or Arg506 and 2) the extent to which it is cleaved initially at Arg306, in contrast to cleavage of Arg506, will define both the extent and rate of inactivation.

Amino Acid Sequence↗
PubMed1995

Characterization of the molecular defect in factor VR506Q.

A poor anticoagulant response of plasma to activated protein C is correlated with a single mutation in the factor V molecule (Arg506-->Gln). Factor V was purified to homogeneity from plasma of two unrelated patients (patient I, factor VI, and patient II, factor VII), who are homozygous for this mutation. The factor V molecule from both patients has normal procoagulant activity when compared with factor V isolated from normal plasma in both a clotting time-based assay and in an assay measuring alpha-thrombin formation. The cleavage and subsequent inactivation by activated protein C (APC) of the alpha-thrombin-activated membrane-bound cofactor (factor Va) from both patients were analyzed and compared with the cleavage and inactivation of normal human factor Va. In normal factor Va, cleavage at Arg506 generates a M(r) = 75,000 fragment and a M(r) = 28,000/26,000 doublet and is necessary for the optimum exposure of the sites for subsequent cleavage at Arg306 and Arg679. Proteolysis at these sites leads to the appearance of M(r) - 45,000 and 30,000 fragments and a M(r) = 22,000/20,000 doublet. Cleavage at Arg306 is membrane-dependent and is required for complete inactivation. Following 5 min of incubation with APC (5.4 nM) membrane-bound normal factor Va (280 nM) has virtually no cofactor activity whereas under similar experimental conditions factor VaI and factor VaII retain approximately 50% of their initial activity. After 1 h of incubation with APC, factor VaI retains 20% of its initial cofactor activity whereas factor VaII has 10% remaining cofactor activity. The initial loss in cofactor activity (approximately 70%) of membrane-bound factor VaI and factor VaII during the first 10 min of the inactivation reaction is correlated with cleavage at Arg306 and appearance of a M(r) = 45,000 fragment and a M(r) = 62,000/60,000 doublet. Subsequently, the M(r) = 62,000/60,000 doublet is cleaved at Arg679 to generate a M(r) = 56,000/54,000 doublet resulting in complete loss of cofactor activity. Both procofactors, factor VI and factor VII, were inactivated following cleavage at Arg306 and Arg679, with APC inactivation rates equivalent to those observed for normal factor V. Our data demonstrate that: 1) cleavage at Arg506 is required for optimum exposure of the cleavage sites at Arg306 and Arg679 and rapid inactivation of membrane-bound factor Va; and 2) cleavage at Arg306 by APC on membrane-bound factor V occurs at the same rate in both normal and APC-resistant individuals. Thus cleavage at Arg306 and Arg679 and subsequent inactivation of the membrane-bound procofactor, factor V, does not require prior cleavage at Arg506 for optimum exposure.

Arginine↗
PubMed1995

Biochemical prototype for familial thrombosis. A study combining a functional protein C mutation and factor V Leiden.

Resistance to activated protein C (APC) is associated with a single amino acid substitution in factor V (Arg506-->Gln, factor V Leiden) that results in delayed inactivation of the molecule by APC. The mutation is present in 20% of patients with a first episode of deep venous thrombosis. Arterial and venous thromboses are also associated with the type II protein C deficiency (protein CVermont). In protein CVermont, the substitution Glu20-->Ala alone (rPC gamma 20A) is responsible for the defective anticoagulant properties of PCVermont. It was recently established that a thrombotic episode occurred in 73% of family members who are heterozygous for both a functional protein C gene mutation and the factor V Leiden mutation. We evaluated the molecular defect that would accrue in the combined deficiency state of factor VR506Q/VaR506Q and rAPC gamma 20A using recombinant APC and natural purified factor VR506Q from patients homozygous for the Arg506-->Gln substitution. While wild-type recombinant APC (rAPC) slowly cleaves and inactivates factor VR506Q and factor VaR506Q, minimal cleavage of membrane-bound factor VR506Q and VaR506Q by rAPC gamma 20A at Arg306 and Arg679 occurs, and no loss in cofactor activity is observed. Our data demonstrate that rAPC gamma 20A cannot inactivate either factor VR506Q or factor VaR506Q at biologically relevant rates because of impaired cleavage at Arg306 and Arg679.(ABSTRACT TRUNCATED AT 250 WORDS)

Factor V↗