Michael Kalafatis

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 ∼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 prothrombinaseactivity 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.

Megakaryocytes endocytose and subsequently modify human factor V in vivo to form the entire pool of a unique platelet‐derived cofactor

Summary.  Factor Va (FVa), derived from plasma or released from stimulated platelets, is the essential cofactor in thrombin production catalyzed by the prothrombinase complex. Plasma‐derived factor V (FV) is synthesized in the liver. The source(s) of the platelet‐derived cofactor remains in question. We identified a patient homozygous for the FVLeiden mutation, who received a liver transplant from a homozygous wild‐type FV donor. Eighteen days post‐transplant, phenotypic analysis of the patients platelet‐derived FV indicated that the platelets were acquiring wild‐type FV, consistent with the temporal differentiation of megakaryocytes and subsequent platelet production. Nine months post‐transplant, the platelet‐derived FV pool consisted entirely of wild‐type FV. Consequently, megakaryocyte endocytosis of plasma‐derived FV must account for the entire platelet‐derived pool, because blood‐borne platelets cannot bind or endocytose FV. Subsequent to this endocytic process, the patients platelet‐derived FV was cleaved to a partially active cofactor, and rendered resistant to phosphorylation catalyzed by a platelet‐associated kinase, and hence less susceptible to activated protein C‐catalyzed inactivation. These data provide the first in vivo demonstration of an endocytosed plasma protein undergoing intracellular modifications that alter its function. This process results in the sequestration of active FVa within the platelet compartment, poised for immediate action subsequent to release from platelets at a site of injury.

Factor VLeiden and Thrombophilia

Blood coagulation is initiated after injury to the vasculature and exposure of TF. TF is an integral single-chain glycoprotein that, when exposed to the circulating blood, binds to circulating factor VIIa and initiates the process of blood coagulation. Although the majority of factor VII molecules circulate in plasma as a single-chain, inactive zymogen, ≈2% of them possess a Ser protease active site but have poor catalytic activity.1 2 After binding to TF, the catalytic efficiency of the factor VIIa/TF complex increases by four orders of magnitude, and the enzymatic complex initiates a series of enzymatic reactions that lead to the generation of α-thrombin.2 3 The prothrombin-activating enzyme prothrombinase is composed of the Ser protease factor Xa and cofactor factor Va, associated on the cell membrane.4 Factor Va is required for prothrombinase activity because it serves the dual function as a factor Xa receptor and a factor Xa catalytic effector on the cell surface. Both enzyme and cofactor are derived from plasma precursors by regulatory proteolytic events that involve α-thrombin. These events include activation of factor VIII to factor VIIIa and of factor V to factor Va. α-Thrombin, the major procoagulant enzyme of the blood coagulation cascade, is also paradoxically a major anticoagulant. Once formed, α-thrombin binds to the endothelial cell receptor thrombomodulin and initiates the protein C pathway that leads to the formation of APC. The protein C pathway is composed of the zymogen protein C, thrombomodulin, and the accessory protein, protein S.5 APC downregulates α-thrombin generation by inactivating factors Va and VIIIa and eliminating their participation in the prothrombinase and tenase complexes, respectively. Under physiological conditions, inactivation of factor VIIIa occurs in the absence of APC by dissociation of the A2 domain.6 7 8 Hence, APC is not essential for factor VIIIa inactivation.8 …

Structural Requirements for Expression of Factor Va Activity

Thrombin activated factor Va (factor VIIa, residues 1–709 and 1546–2196) has an apparent dissociation constant (Kd,app) for factor Xa within prothrombinase of ∼0.5 nm. A protease (NN) purified from the venom of the snake Naja nigricollis nigricollis, cleaves human factor V at Asp697, Asp1509, and Asp1514 to produce a molecule (factor VNN) that is composed of a Mr 100,000 heavy chain (amino acid residues 1–696) and a Mr 80,000 light chain (amino acid residues 1509/1514–2196). Factor VNN, has a Kd,app for factor Xa of 4 nm and reduced clotting activity. Cleavage of factor VIIa by NN at Asp697 results in a cofactor that loses ∼60–80% of its clotting activity. An enzyme from Russells viper venom (RVV) cleaves human factor V at Arg1018 and Arg1545 to produce a Mr 150,000 heavy chain and Mr 74,000 light chain (factor VRVV, residues 1–1018 and 1546–2196). The RVV species has affinity for factor Xa and clotting activity similar to the thrombin-activated factor Va. Cleavage of factor VNN at Arg1545 by α-thrombin (factor VNN/IIa) or RVV (factor VNN/RVV) leads to enhanced affinity of the cofactor for factor Xa (Kd,app ∼ 0.5 nm). A synthetic peptide containing the last 13 residues from the heavy chain of factor Va (amino acid sequence 697–709, D13R) was found to be a competitive inhibitor of prothrombinase with respect to prothrombin. The peptide was also found to specifically interact with thrombin-agarose. These data demonstrate that 1) cleavage at Arg1545 and formation of the light chain of factor VIIa is essential for high affinity binding and function of factor Xa within prothrombinase and 2) a binding site for prothrombin is contributed by amino acid residues 697–709 of the heavy chain of the cofactor.

The contribution of amino acid region Asp695-Tyr698 of factor V to procofactor activation and factor Va function

There is strong evidence that a functionally important cluster of amino acids is located on the COOH-terminal portion of the heavy chain of factor Va, between amino acid residues 680 and 709. To ascertain the importance of this region for cofactor activity, we have synthesized five overlapping peptides representing this amino acid stretch (10 amino acids each, HC1-HC5) and tested them for inhibition of prothrombinase assembly and function. Two peptides, HC3 (spanning amino acid region 690-699) and HC4 (containing amino acid residues 695-704), were found to be potent inhibitors of prothrombinase activity with IC50 values of ∼12 and ∼10 μm, respectively. The two peptides were unable to interfere with the binding of factor Va to active site fluorescently labeled Glu-Gly-Arg human factor Xa, and kinetic analyses showed that HC3 and HC4 are competitive inhibitors of prothrombinase with respect to prothrombin with Ki values of ∼6.3 and ∼5.3 μm, respectively. These data suggest that the peptides inhibit prothrombinase because they interfere with the incorporation of prothrombin into prothrombinase. The shared amino acid motif between HC3 and HC4 is composed of Asp695-Tyr-Asp-Tyr-Gln699 (DYDYQ). A pentapeptide with this sequence inhibited both prothrombinase function with an IC50 of 1.6 μm (with a KD for prothrombin of 850 nm), and activation of factor V by thrombin. Peptides HC3, HC4, and DYDYQ were also found to interact with immobilized thrombin. A recombinant factor V molecule with the mutations Asp695 → Lys, Tyr696 → Phe, Asp697 → Lys, and Tyr698 → Phe (factor V2K2F) was partially resistant to activation by thrombin but could be readily activated by RVV-V activator (factor VaRVV2K2F) and factor Xa (factor VaXa2K2F). Factor VaRVV2K2F and factor VaXa2K2F had impaired cofactor activity within prothrombinase in a system using purified reagents. Our data demonstrate for the first time that amino acid sequence 695-698 of factor Va heavy chain is important for procofactor activation and is required for optimum prothrombinase function. These data provide functional evidence for an essential and productive contribution of factor Va to the activity of prothrombinase.

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.

The Structural Integrity of Anion Binding Exosite I of Thrombin Is Required and Sufficient for Timely Cleavage and Activation of Factor V and Factor VIII

α-Thrombin has two separate electropositive binding exosites (anion binding exosite I, ABE-I and anion binding exosite II, ABE-II) that are involved in substrate tethering necessary for efficient catalysis. α-Thrombin catalyzes the activation of factor V and factor VIII following discrete proteolytic cleavages. Requirement for both anion binding exosites of the enzyme has been suggested for the activation of both procofactors by α-thrombin. We have used plasma-derived α-thrombin, β-thrombin (a thrombin molecule that has only ABE-II available), and a recombinant prothrombin molecule rMZ-II (R155A/R284A/R271A) that can only be cleaved at Arg320 (resulting in an enzymatically active molecule that has only ABE-I exposed, rMZ-IIa) to ascertain the role of each exosite for procofactor activation. We have also employed a synthetic sulfated pentapeptide (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{DY}(\mathrm{SO}_{3}^{-})\mathrm{DY}(\mathrm{SO}_{3}^{-})\mathrm{Q}\) \end{document}, designated D5Q1,2) as an exosite-directed inhibitor of thrombin. The clotting time obtained with β-thrombin was increased by ∼8-fold, whereas rMZ-IIa was 4-fold less efficient in promoting clotting than α-thrombin under similar experimental conditions. α-Thrombin readily activated factor V following cleavages at Arg709, Arg1018, and Arg1545 and factor VIII following proteolysis at Arg372, Arg740, and Arg1689. Cleavage of both procofactors byα-thrombin was significantly inhibited by D5Q1,2. In contrast, β-thrombin was unable to cleave factor V at Arg1545 and factor VIII at both Arg372 and Arg1689. The former is required for light chain formation and expression of optimum factor Va cofactor activity, whereas the latter two cleavages are a prerequisite for expression of factor VIIIa cofactor activity. β-Thrombin was found to cleave factor V at Arg709 and factor VIII at Arg740, albeit less efficiently than α-thrombin. The sulfated pentapeptide inhibited moderately both cleavages by β-thrombin. Under similar experimental conditions, membrane-bound rMZ-IIa cleaved and activated both procofactor molecules. Activation of the two procofactors by membrane-bound rMZ-IIa was severely impaired by D5Q1,2. Overall the data demonstrate that ABE-I alone of α-thrombin can account for the interaction of both procofactors with α-thrombin resulting in their timely and efficient activation. Because formation of meizothrombin precedes that of α-thrombin, our findings also imply that meizothrombin may be the physiological activator of both procofactors in vivo in the presence of a procoagulant membrane surface during the early stages of coagulation.

[13] Factor V

Publisher Summary The proteolytic activation of prothrombin is catalyzed by an enzyme complex composed of the serine protease factor Xa reversibly associated with the cofactor factor Va on membranes containing negatively charged phospholipid in the presence of calcium ions. Studies of the structure–function relationships in factor Va have been aided by the availability of the sequence of the cDNA coding for factor V by the development of binding and kinetic measurements to examine individually the discrete interactions of the cofactor within prothrombinase and by the isolation and characterization of proteolytic fragments through protein chemistry techniques. This chapter discusses the methodology for the isolation of factor Va, its subunits, and proteolytic derivatives of the cofactor and describes the various steps of the purification and partial characterization of a small region of the Va LC of the cofactor, which possess a phospholipid binding domain. The chapter describes the enzymatic degradation of Va LC in presence of phospholipid vesicles.

A Control Switch for Prothrombinase CHARACTERIZATION OF A HIRUDIN-LIKE PENTAPEPTIDE FROM THE COOH TERMINUS OF FACTOR Va HEAVY CHAIN THAT REGULATES THE RATE AND PATHWAY FOR PROTHROMBIN ACTIVATION

Membrane-bound factor Xa alone catalyzes prothrombin activation following initial cleavage at Arg271 and prethrombin 2 formation (pre2 pathway). Factor Va directs prothrombin activation by factor Xa through the meizothrombin pathway, characterized by initial cleavage at Arg320 (meizo pathway). We have shown previously that a pentapeptide encompassing amino acid sequence 695–699 from the COOH terminus of the heavy chain of factor Va (Asp-Tyr-Asp-Tyr-Gln, DYDYQ) inhibits prothrombin activation by prothrombinase in a competitive manner with respect to substrate. To understand the mechanism of inhibition of thrombin formation by DYDYQ, we have studied prothrombin activation by gel electrophoresis. Titration of plasma-derived prothrombin activation by prothrombinase, with increasing concentrations of peptide, resulted in complete inhibition of the meizo pathway. However, thrombin formation still occurred through the pre2 pathway. These data demonstrate that the peptide preferentially inhibits initial cleavage of prothrombin by prothrombinase at Arg320. These findings were corroborated by studying the activation of recombinant mutant prothrombin molecules rMZ-II (R155A/R284A/R271A) and rP2-II (R155A/R284A/R320A) which can be only cleaved at Arg320 and Arg271, respectively. Cleavage of rMZ-II by prothrombinase was completely inhibited by low concentrations of DYDYQ, whereas high concentrations of pentapeptide were required to inhibit cleavage of rP2-II. The pentapeptide also interfered with prothrombin cleavage by membrane-bound factor Xa alone in the absence of factor Va increasing the rate for cleavage at Arg271 of plasma-derived prothrombin or rP2-II. Our data demonstrate that pentapeptide DYDYQ has opposing effects on membrane-bound factor Xa for prothrombin cleavage, depending on the incorporation of factor Va in prothrombinase.

The role of the membrane in the inactivation of factor va by plasmin. Amino acid region 307-348 of factor V plays a critical role in factor Va cofactor function.

The mechanism of inactivation of bovine factor Va by plasmin was studied in the presence and absence of phospholipid vesicles (PCPS vesicles). Following 60-min incubation with plasmin (4 nm) membrane-bound factor Va (400 nm) is completely inactive, whereas in the absence of phospholipid vesicles following a 1-h incubation period, the cofactor retains 90% of its initial cofactor activity. Amino acid sequencing of the fragments deriving from cleavage of factor Va by plasmin demonstrated that while both chains of factor Va are cleaved by plasmin, only cleavage of the heavy chain correlates with inactivation of the cofactor. In the presence of a membrane surface the heavy chain of the bovine cofactor is first cleaved at Arg348 to generate a fragment of M r 47,000 containing the NH2-terminal part of the cofactor (amino acid residues 1–348) and a M r 42,000 fragment (amino acid residues 349–713). This cleavage is associated with minimal loss in cofactor activity. Complete loss of activity of the membrane-bound cofactor coincides with three cleavages at the COOH-terminal portion of the M r 47,000 fragment: Lys309, Lys310, and Arg313. These cleavages result in the release of the COOH terminus of the molecule and the production of a M r 40,000 fragment containing the NH2-terminal portion of the factor Va molecule. Factor Va was treated with plasmin in the absence of phospholipid vesicles followed by the addition of PCPS vesicles and activated protein C (APC). A rapid inactivation of the cofactor was observed as a result of cleavage of the M r 47,000 fragment at Arg306 by APC and appearance of aM r 39,000 fragment. These data suggest a critical role of the amino acid sequence 307–348 of factor Va. A 42-amino acid peptide encompassing the region 307–348 of human factor Va (N42R) was found to be a good inhibitor of factor Va clotting activity with an IC50 of ∼1.3 μm. These data suggest that plasmin is a potent inactivator of factor Va and that region 307–348 of the cofactor plays a critical role in cofactor function and may be responsible for the interaction of the cofactor with factor Xa and/or prothrombin.