Plasma Factor Xa Research Articles

Monitoring Anticoagulation Therapy in Patients with the Lupus Anticoagulant

Patients with a lupus anticoagulant (LAC) may present with thrombosis, and abnormal coagulation studies. It is important to recognize that these individuals may require special laboratory tests other than measuring the activated partial thromboplastin time or prothrombin time/international normalized ratio (PT/INR) traditionally used by clinicians to monitor hep-arin and warfarin. We recommend the use of a factor Xa inhibition test to regulate heparin in this patient population. This test measures the ability of heparin, as a cofactor of antithrombin III, to inMbit the catalytic function of factor Xa in plasma. Therapeutic levels are 0.3-0.7 U/mL of anti-Xa activity. For warfarin, we suggest measuring the factor X levels as determined by a chromogenic assay, if the patient initially has a prolonged PT/ INR. Therapeutic levels are 10%-40% of normal values. In this article, we review this and other tests available to monitor antithrornbotic therapy in patients with the LAC.

Binding sites for blood coagulation factor Xa and protein S involving residues 493-506 in factor Va.

Inactivation due to cleavage of Factor Va (FVa) at Arg 506 by activated protein C (APC) helps to downregulate blood coagulation. To identify potential functional roles of amino acids near Arg 506, synthetic overlapping pentadecapeptides comprising FVa heavy chain residues 481-525 were tested for their ability to inhibit prothrombin activation by prothrombinase complexes [Factor Xa (FXa):FVa:phospholipids:Ca2+]. The most potent inhibition was observed for peptide VP493 (residues 493-506), with 50% inhibition at 2.5 microM. VP493 also inhibited FXa in plasma in FXa-1-stage clotting assays by 50% at 3 microM. When the C-terminal carboxamide group of VP493 was replaced by a carboxyl group, most prothrombinase inhibitory activity was lost. VP493 preincubated with FXa inhibited prothrombinase with a pattern of mixed inhibition. Homologous peptides from Factor VIII sequences did not inhibit prothrombinase. Affinity-purified antibodies to VP493 inhibited prothrombinase activity and prolonged FXa-1-stage clotting times. VP493 also blocked the ability of protein S to inhibit prothrombinase independently of APC. Immobilized VP493 bound specifically with similar affinity to both FXa and protein S (Kd approximately 40 nM), but did not measurably bind prothrombin or APC. These studies suggest that FVa residues 493-506 contribute to binding sites for both FXa and protein S, providing a rationale for the ability of protein S to negate the protective effect of FXa toward APC cleavage of FVa. Possible loss of this FVa binding site for FXa due to cleavage at Arg 506 by APC may help explain why this cleavage causes 40% decrease in FVa activity and facilitates inactivation of FVa.

Procoagulant Activity of Reversibly Acylated Human Factor Xa

The plasma clotting factors used to treat hemophiliacs who have developed inhibitory antibodies have a shared history of limited clinical safety and utility. To improve on existing bypass factors, we have developed a reversibly acylated form of human plasma factor Xa capable of providing a time-dependent release of procoagulant activity. Factor Xa was treated with p-amidinophenyl p'-anisate to generate anisoyl Xa. The chemical modification of the protein involves acylation of the active site serine residue of factor Xa. Anisoyl Xa deacylated in a time, pH, and temperature-dependent manner. Active factor Xa generated on deacylation of anisoyl Xa exhibited amidolytic and prothrombinase complex activities in in vitro assays, the level being comparable to those of untreated factor Xa. When Anisoyl Xa was infused into rabbits, active factor Xa was generated on deacylation of the acylated enzyme, which shortened the activated partial thromboplastin time (APTT) in a dose-dependent manner. The duration of effect on rabbit APTT could be directly correlated to the level of human plasma factor Xa. Because anisoyl Xa bypasses the "tenase" complex that is compromised in hemophilia A and B and is unaffected by inhibitory antibodies, it has the potential to be used as an effective bypass therapy.

Effect of gamma carboxylation on prothrombinase inhibitory activity of catalytically inactive factor Xa

Recombinant catalytically inactive factor Xa (factor rXai) is capable of assembly into inactive prothrombinase complexes, thus serving as a competitive inhibitor (Ki = 0.3nM) of active factor Xa. In order to study the role of gamma carboxylation in prothrombinase complex assembly, we have prepared differentially gamma carboxylated factor rXai and have measured the activities of these proteins in prothrombinase complex inhibition and in extension of plasma clotting. A factor rXai preparation containing 8 out of a possible maximum of 11 g carboxyglutamic acid (GLA) residues was found to be as active as chemically inactivated plasma factor Xa which was fully gamma carboxylated. Loss of a single additional g carboxyglutamic acid in the recombinant protein, however lead to a marked loss in activity. Factor rXai preparation with 8 GLA residues is also detected by a monoclonal antibody specific for a GLA dependent epitope. Thus assembly of the factor Va/Xa complex on phospholipid membranes does not require the presence of all of the g carboxyglutamic acid residues present in the plasma protein.

In vivo pharmacology of aprosulate, a new synthetic polyanion with anticoagulant activity

The in vivo effects of a new synthetic inhibitor of blood coagulation, aprosulate sodium, were investigated. Intravenous bolus injection of aprosulate or standard heparin in rats produced an immediate prolongation of the APTT which were characterized by a moderate dose-dependency and long-lasting duration when compared with those of standard heparin. Standard heparin inhibited plasma factor Xa activity, but aprosulate did not even at the highest dose used. Both agents inhibited thrombus formation in a dose-dependent manner in an arterio-venous shunt model. At antithrombotic doses, standard heparin prolonged the bleeding time measured by the tail transection method, but aprosulate did not. The present results suggest that aprosulate has promising in vivo profile as an anticoagulant and antithrombotic agent.

Expression, purification, and characterization of inactive human coagulation factor Xa (Asn 322Ala 419)

We have expressed in Chinese hamster ovary cells a catalytically inactive form of human factor Xa (factor rXai). A recombinant precursor of human factor Xa was inactivated by two point mutations in the serine protease catalytic triad, Asp322Asn and Ser419Ala. A two-step purification to homogeneity of the secreted material involved immunoaffinity followed by heparin-agarose chromatography. Two forms were identified; a fully processed dimer (70%) and a partially processed monomer (30%). Limited N-terminal amino acid sequencing of factor rXai detected the predicted residues and γ-carboxyglutamic acid content was 90% of human plasma control. Although devoid of measurable proteolytic activity, factor rXai competitively inhibited plasma factor Xa assembly into functional prothrombinase complexes ( K i = 3 × 10 −10 m). Factor rXai also inhibited plasma clotting in a dose-dependent manner. The possible use of recombinant catalytically inactive proteins as a general approach for pharmacological regulation of human diseases is discussed.

Congenital antithrombin III deficiency: insights into the pathogenesis of the hypercoagulable state and its management using markers of hemostatic system activation

Hereditary antithrombin III (ATIII) deficiency predisposes patients to venous thrombosis. The prothrombin fragment F1+2 radioimmunoassay demonstrates that many asymptomatic patients with this disorder not receiving antithrombotic therapy have elevated plasma factor Xa activity. The hemostatic system hyperactivity as measured by this assay could be specifically corrected by rising plasma ATIII levels of several persons into the normal range. This indicates that the prethrombotic state can be defined as an imbalance between the production and inhibition of factor Xa enzymatic activity. The effects of warfarin on factor Xa enzymatic activity in persons with congenital ATIII deficiency have also been evaluated. At equivalent intensities of oral anticoagulation, the mean plasma F1+2 level in patients with ATIII deficiency was significantly elevated as compared with anticoagulated persons without this inherited thrombotic disorder. It is concluded that the effect of warfarin on hemostatic system activation is modulated by the endogenous heparan sulfate-ATIII mechanism. This suggests that the F1+2 radioimmunoassay can be employed to improve the understanding of the hypercoagulable state associated with antithrombin III deficiency as well as to develop more effective treatment strategies to prevent thromboembolic events in patients with this disorder.

Electrochemical activity determination of trypsin-like enzymes. IX. Factor X and factor Xa in plasma and whole blood

Electrochemical activity determination of trypsin-like enzymes. IX. Factor X and factor Xa in plasma and whole blood

Heparan sulfate and dermatan sulfate inhibit the generation of thrombin activity in plasma by complementary pathways

Heparan with a low affinity for antithrombin III has previously been demonstrated to inhibit thrombin generation in both normal plasma and plasma depleted of antithrombin III. In addition, standard heparin and heparin with a low affinity for antithrombin III have been demonstrated to have equivalent inhibitory actions on thrombin generation in plasma depleted of antithrombin III. These observations prompted the investigation of the effects of four normal vessel wall glycosaminoglycans (heparan sulfate, dermatan sulfate, chondroitin-4- sulfate, and chondroitin-6-sulfate) on the intrinsic pathway generation of thrombin and factor Xa and on the inactivation of thrombin and factor Xa in plasma. Heparan sulfate inhibited thrombin generation and accelerated the inactivation of added thrombin and factor Xa in normal plasma but not in antithrombin III-depleted plasma. In contrast, dermatan sulfate inhibited thrombin generation in both normal and antithrombin III-depleted plasma. In addition, heparan sulfate was an effective inhibitor of factor Xa generation, while dermatan sulfate was not. Neither chondroitin-4-sulfate nor chondroitin-6-sulfate inhibited the generation of thrombin or factor Xa nor did they accelerate the inactivation of factor Xa or thrombin by plasma. These results suggest that heparan sulfate acts primarily by potentiating antithrombin III, while dermatan sulfate acts by potentiating heparin cofactor II. The inhibition of thrombin generation by heparan sulfate and dermatan sulfate thus appears to occur by complementary pathways, both of which may contribute to the anticoagulation of blood in vivo.

Heparan Sulfate and Dermatan Sulfate Inhibit the Generation of Thrombin Activity in Plasma by Complementary Pathways

Heparan with a low affinity for antithrombin III has previously been demonstrated to inhibit thrombin generation in both normal plasma and plasma depleted of antithrombin III. In addition, standard heparin and heparin with a low affinity for antithrombin III have been demonstrated to have equivalent inhibitory actions on thrombin generation in plasma depleted of antithrombin III. These observations prompted the investigation of the effects of four normal vessel wall glycosaminoglycans (heparan sulfate, dermatan sulfate, chondroitin-4-sulfate, and chondroitin-6-sulfate) on the intrinsic pathway generation of thrombin and factor Xa and on the inactivation of thrombin and factor Xa in plasma. Heparan sulfate inhibited thrombin generation and accelerated the inactivation of added thrombin and factor Xa in normal plasma but not in antithrombin III-depleted plasma. In contrast, dermatan sulfate inhibited thrombin generation in both normal and antithrombin III-depleted plasma. In addition, heparan sulfate was an effective inhibitor of factor Xa generation, while dermatan sulfate was not. Neither chondroitin-4-sulfate nor chondroitin-6-sulfate inhibited the generation of thrombin or factor Xa nor did they accelerate the inactivation of factor Xa or thrombin by plasma. These results suggest that heparan sulfate acts primarily by potentiating antithrombin III, while dermatan sulfate acts by potentiating heparin cofactor II. The inhibition of thrombin generation by heparan sulfate and dermatan sulfate thus appears to occur by complementary pathways, both of which may contribute to the anticoagulation of blood in vivo.

GAGs-Potentiated Inhibition of Thrombin, Factor Xa and Plasmin in Plasma and in a Purified System Containing Antithrombin III – Correlation with Total Charge Density

The ability of heparin and related glycosaminoglycans (GAGs) to accelerate the inhibition of thrombin, factor Xa and plasmin in plasma and in a purified system containing antithrombin III (At III) was studied using chromogenic peptide substrate assays. There was good correlation between the charge density of the mucopolysaccharides and the activities investigated. While the difference between potentiation of the antithrombin activity by GAGs in plasma and in the purified system was slight, the inhibition of factor Xa in plasma was more pronounced than in the presence of purified At III, indicating the mechanisms for GAGs-potentiated inhibition of thrombin and factor Xa are not identical. For the antiplasmin activity, there was a good correlation between the chemical structure and biological activity only in the pure system, confirming that the antithrombin-GAG complex plays a very limited role in the inactivation of plasmin in plasma.

Molecular weight dependency of the heparin potentiated inhibition of thrombin and activated factor X. Effect of heparin neutralization in plasma

Heparin purified by affinity chromatography on antithrombin III-Sepharose was fractionated according to molecular size by gel filtration. The various fractions obtained were studied with respect to their ability to potentiate the inhibition of thrombin and of Factor Xa in plasma and in a purified system containing antithrombin III. Clotting assays and chromogenic peptide substrate assays were used in parallel. The inhibition of thrombin showed a dependency on the molecular size of the heparin that was similar to that shown earlier for the APTT assay. The inhibition of Factor Xa showed a different dependency, indicating that the mechanisms for heparin-potentiated inhibition of thrombin and Factor Xa by antithrombin III are not entirely the same. The inhibition of thrombin and Factor Xa in plasma differed from that in the pure system and this difference was shown to be dependent on heparin neutralization effects.

The Effect of Antithrombin III on the Activity of the Coagulation Factors VII, IX and X

Antithrombin III, purified to homogeneity according to polyacrylamide gel disc electrophoresis and immunoelectrophoresis, inhibited the activity of purified factor IXa and Xa, whereas factor VII was not inhibited either in the active or in the native form. Antithrombin III is the single most important inhibitor of factor Xa in plasma. Factor Xa does not, however, reduce the activity of antithrombin III against thrombin.

Activation of prothrombin with (taipan snake) venom

Complete activation of prothrombin with taipan snake () venom yields thrombin and two fragments, fragment-1 and fragment-2, which by the criteria of ion-exchange chromatography and SDS gel electrophoresis are indistinguishable from the products of Xa-catalyzed prothrombin activation. Activation in the presence of diisopropylfluorophosphate with either taipan venom or Xa results in the formation of thrombin-sensitive fragment 1·2, a single polypeptide composed of fragments-1 and 2. Whereas the rate of venom-catalyzed activation is enhanced 20–30 times by phospholipid vesicles, Factor V is without effect in either the presence or absence of phospholipid. The venom activity is not inhibited by diisopropylfluorophosphate, phenylmethylsulfonylfluoride, soybean trypsin inhibitor, p-hydroxymercuribenzoate or iodoacetate. The venom does not hydrolyze N2-toluene-p-sulfonyl-L-arginine methyl ester, N2-benzoyl-L-arginine ethyl ester or N-acetyl-L-tyrosine ethyl ester. The rate of venom-catalyzed prothrombin activation is not affected by plasma factor Xa inhibitor plus heparin.