scholarly journals The Action of a Synthetic Pentasaccharide on Thrombin Generation in Whole Plasma

1989 ◽  
Vol 61 (03) ◽  
pp. 397-401 ◽  
Author(s):  
S Béguin ◽  
J Choay ◽  
H C Hemker
Keyword(s):  
Binding Site ◽  
Blood Coagulation ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Factor Xa ◽  
Heparin Binding ◽  
Extrinsic Pathway ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Prothrombinase Activity

SummaryWe investigated the effect on thrombin generation in plasma of the pentasaccharide that represent the AT II/binding site in heparin. This compound has no effect on the breakdown of thrombin in plasma. It dose-dependently inhibits the formation of thrombin in both the intrinsic and the extrinsic pathway. If coagulation is triggered by the complete prothrombinase complex (phospholipid – factor Va – factor Xa) under conditions in which the large majority of factor Xa is bound to the complex, the inhibition of prothrombinase activity is only minor. If no factor Va is present or if the prothrombinase activity is triggered by adding complete tenase (PL-FVIIIa-FIXa) or incomplete tenase (PLFIXa) to the plasma the inhibition by pentasaccharide is of the same magnitude as that in the intrinsic or extrinsic system.We conclude that the pentasaccharide inhibits blood coagulation by katalysing the inactivation of free factor Xa. In contrast to classical heparin it does inhibit the peak of thrombin formation in platelet rich plasma, probably because it is less subject to inactivation by heparin binding proteins from platelets than classical heparin is.

Abstract 30: Tissue Factor Pathway Inhibitor α Inhibits Prothrombinase via a Three-Step Mechanism

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Jeremy P Wood ◽  
Alan E Mast
Keyword(s):  
Active Site ◽  
Inhibitory Activity ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Factor Xa ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Direct Binding ◽  
Affinity Interaction

TFPIα inhibits early forms of the prothrombinase complex (factor Xa (FXa), factor Va (FVa)), though the inhibitory mechanism is not entirely understood. One step of inhibition is a high affinity interaction between a TFPIα C-terminal basic region (BR) (252-LIKTKRKRKK-261) and an acidic region (AR) present in FXa-activated and platelet-released forms of FVa. We investigated two additional potential mechanistic steps: (1) binding of the second Kunitz-type inhibitory domain (K2) of TFPIα to the FXa active site; and (2) the function of uncharged residues L252, I253, and T255 within the BR, which are evolutionarily conserved, suggesting they have activity. Direct inhibition of FXa was investigated using TFPIα with an altered K2 (TFPI-R107A) incapable of binding FXa. TFPI-R107A inhibited purified prothrombinase 17-fold weaker than TFPIα (IC50 = 30.6nM vs. 1.8nM) and did not inhibit FXa-initiated thrombin generation in platelet-rich plasma (PRP). Therefore, direct binding of FXa and K2 is required for efficient inhibition of prothrombinase under physiological conditions. Similarly, the role of L252, I253, and T255 was investigated by substituting them with alanine (TFPI-AAKA). The IC50 for prothrombinase inhibition by TFPI-AAKA was 10.4nM, and it had reduced inhibitory activity in PRP, revealing that these residues are also required for efficient prothrombinase inhibition. The role of L252, I253, and T255 was further probed using the peptide LIKTKRKRKK, which inhibited purified prothrombinase (IC50 = 1.0μM) and thrombin generation in PRP at 1μM. AAKAKRKRKK had very little activity in either assay (~20% prothrombinase inhibition with 225μM peptide), but bound the FVa AR equivalently to LIKTKRKRKK (K d = 5.9nM and 6.0nM, respectively). Thus, the basic residues are responsible for AR binding, while a second step, mediated by L252-T255, is necessary for inhibitory activity. These residues may be necessary for displacement of FXa from FVa, as proposed by Bunce et al. We propose that prothrombinase inhibition by TFPIα involves three steps: (1) the TFPIα BR basic residues bind the FVa AR; (2) residues L252-T255 block prothrombinase assembly; and (3) K2 binds the FXa active site. All three steps are required for physiologic inhibition of prothrombinase by TFPIα.

Stressed Endothelial Cells Compartmentalize the Prothrombinase Complex on Filopodia and Other Convex Membrane Features near the Cell Margin

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 372-372
Author(s):  
Jialan Shi ◽  
Dessislava N. Nikova ◽  
Gary E. Gilbert
Keyword(s):  
Endothelial Cells ◽  
Binding Sites ◽  
Umbilical Vein ◽  
Factor Xa ◽  
Annexin V ◽  
Complex Assembly ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Tnf Α ◽  
Prothrombinase Activity

Abstract Abstract 372 The dependence of procoagulant activity on phosphatidylserine (PS) has been recognized for at least four decades but the location of physiologically relevant membranes with PS exposure remains uncertain. PS is exposed on apoptotic cells and cell microparticles but in vitro and in vivo studies have failed to demonstrate a clear relationship of microparticles or apoptotic cells to fibrin deposition. Exposure of endothelial cells to stimulants or toxins leads to retraction of cell margins, mounding of the central cell, and extension of filopodia. We have also found that cell stress also leads to limited, focal PS exposure. Furthermore, we found that binding sites for lactadherin, a PS-binding protein that shares homology with factor VIII and factor V, are concentrated on convex surfaces such as filopodia. In this study we ask whether the limited, focal PS exposure on stressed human umbilical vein endothelial cells is sufficient to support prothrombinase complex assembly and whether the prothrombinase complex assembly is restricted to the convex membrane features that bind lactadherin. We allowed Human Umbilical Vein Endothelial Cells (HUVEC) to grow to confluent monolayers prior to exposure to TNF-α, 10 ng/ml, for 5–24 hours. PS exposure was detected by simultaneous staining using 10 nM lactadherin–Alexa 488 and annexin V–Cy 3.18, both exhibiting high affinity for PS. Stressed cells withdrew from their prior borders, leaving residual fibrils connected to original attachment points. In addition, they extended filopodia that were up to several cell diameters in length. Confocal microscopy demonstrated focal staining of filopodia, fibrils and cell margins with lactadherin and patches near the nucleus with annexin A5. We asked whether the selective binding might be determined by the membrane topology. To mimic the curvature of a cell membrane we prepared nano-fabricated silica substrates with ridge radii of 10 nm. The AFM topographic and fluorescent images of synthetic membrane bilayers supported by the substrates showed that, over a PS content of 4–15%, lactadherin preferentially binds to the convex nano-ridges with a ridge: valley staining ratio >80:1, while annexin V selectively binds the concave areas of the nano-trenches with a ridge. Combined fluorescence/AFM imaging of TNF-α treated HUVEC's, demonstrated that the new thin filaments staining with lactadherin had radii of curvature of approx. 12 nm, similar to the ridges of our synthetic bilayers. We asked whether factor Va and factor Xa share preference for convex surfaces, analogous to lactadherin. Supported membranes of 4% PS had preferential ridge staining by factor Va-fluorescein-maleimide with a ridge/valley ratio > 10/1. Co-staining with factor Va and factor Xa-EGRck-biotin (complexed to Alexa 647-steptavidin) indicated that factor Va enhanced binding of factor Xa to ridges, thus the prothrombinase complex has highly preferential binding to convex ridges. TNF-α-treated endothelial cells bound factor Va, like lactadherin, selectively on filopodia and fibrils near the retracted edges of endothelial cells. Factor Xa also localized to these features in the presence of factor Va, indicating prothrombinase complex assembly. Stressed endothelial cells exhibited at least 8-fold higher support for thrombin production and prothrombinase activity. Prothrombinase activity was efficiently inhibited by lactadherin, demonstrating that the lactadherin-binding sites were the functional sites for prothrombinase activity. Together, these data indicate that stressed endothelial cells can support the prothrombinase complex and that prothrombinase activity is compartmentalized near the periphery of the cell and in the intracellular area through binding sites on highly convex membrane features with exposed PS. We have hypothesized that this compartment of procoagulant activity is relatively protected from anti-coagulant proteins that are localized elsewhere on the stimulated/stressed endothelial cell. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Endotoxin enhances the expression of monocyte prothrombinase activity

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 406-416 ◽  
Author(s):  
RA Robinson ◽  
L Worfolk ◽  
PB Tracy
Keyword(s):  
Mononuclear Cells ◽  
Membrane Surface ◽  
Factor Xa ◽  
Dependent Manner ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Flow Cytometric ◽  
And Function ◽  
Microscopy Techniques ◽  
Prothrombinase Activity

Thrombin is generated on the surface of mononuclear cells (MNCs) through the assembly and function of the prothrombinase complex consisting of the enzyme factor Xa, the cofactor/factor Va, calcium ions, and an appropriate membrane surface for proper assembly of the protein constituents. Assays performed in the presence of factors Va and Xa indicated that endotoxin significantly enhanced the prothrombinase activity (1.5- to 2.5-fold; P less than .001) expressed by MNCs in a dose- and time-dependent manner. Monocytes present in the MNC suspensions were responsible for this increased activity through processes resulting in both enhanced cellular activity and the enhanced release of membranous vesicles. Endotoxin was without effect on the expression of lymphocyte prothrombinase activity. Scanning electron microscopy techniques indicated that endotoxin resulted in extensive membrane blebbing of the monocytes present in the MNC suspensions with no effect on the morphology of the lymphocytes. Within 5 hours, endotoxin maximally enhanced the prothrombinase activity expressed by the monocyte membrane surface 2.8-fold, whereas 8 hours was required to maximally enhance the activity associated with the released vesicles by twofold. The observed increase in activity expressed by the monocyte membrane surface was due solely to endotoxin, since the activity expressed by the unstimulated monocyte membrane surface remained unaltered over time. In contrast, cell vesiculation, which occurred in the absence of any stimulus, was further enhanced by endotoxin. The increase in activity associated with the released vesicles from both stimulated and unstimulated cells paralleled an increase in the vesicle number as determined by flow cytometric analyses. The vesicle released from both unstimulated and stimulated monocytes were indistinguishable in size as determined by image analysis and ranged between 0.05 and 0.3 microns in diameter. 2-Deoxy-D-glucose (2DG) significantly enhanced the prothrombinase activity expressed by the monocyte membrane surface, as well as the released vesicle fraction, when used alone or in addition to endotoxin. The enhanced activity associated with the vesicle fraction again was attributed to the release of more vesicles. In contrast, cycloheximide decreased the prothrombinase activity expressed by the monocyte membrane surface, as well as the activity associated with vesicles released from both stimulated and unstimulated cells. These data suggest that the expression of monocyte prothrombinase activity can be significantly enhanced by endotoxin through processes that alter the monocyte membrane surface and augment the vesiculation process. Both processes appear to be regulated by protein synthesis and adenosine triphosphate (ATP)-dependent mechanisms.

Cholesterol and Phosphatidylethanolamine Enhance the Binding of Factor Va and Factor Xa to Phospholipid Vesicles and Promote Assembly of the Prothrombinase Complex on Membranes Containing Low Concentrations of Phosphatidylserine.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1712-1712
Author(s):  
Xiaoe Liang ◽  
Mary Ann Quinn-Allen ◽  
William H. Kane
Keyword(s):  
Phospholipid Composition ◽  
Factor Xa ◽  
Phospholipid Vesicles ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Activated Platelets ◽  
Low Concentrations ◽  
Kd Values ◽  
Direct Binding ◽  
Prothrombinase Activity

Abstract Platelet membranes are composed of cholesterol (CH) and phospholipids including phosphatidylethanolamine (PE), sphingomeylin (SM), phosphatidylcholine (PC) and phosphatidylserine (PS). Phospholipid vesicles containing 20–25% PS have been used extensively to characterize assembly and regulation of the prothrombinase complex. However, the concentration of PS on the surface of activated platelets has been determined to be only 4–13%. Smeets et al (Thromb. Res. 81:419, 1996) demonstrated that CH and PE in the presence of 0–10% PS have stimulating effects on prothrombinase activity, while SM has an inhibitory one. To further investigate the roles of CH, PE and SM in the binding of factor Va and Xa to phospholipid vesicles and assembly of the prothrombinase complex, vesicles consisting of 1–25% DOPS plus 30% CH, 30% DOPE and/or 20% SM, were prepared for prothrombinase assays as a function of DOPS concentration. Compared to individual effect of DOPS alone or DOPS plus CH, PE or SM on prothrombinase activity, a synergistic effect was observed on vesicles containing CH, DOPE and SM, which was significant in the range of 1–15% DOPS. The PS concentration required for half-maximal rates of thrombin generation was 5.3% for vesicles containing CH, DOPE and SM, compared to 10.6, 11.2, 8.3 and 12.6% for vesicles containing DOPS alone, plus CH, plus DOPE or plus SM, respectively. This demonstrates that the requirement for PS is substantially decreased in the presence of CH, PE and SM. In order to further define the mechanisms for this effect, direct binding of factor Va to vesicles containing 30% CH, 30% PE and 20% SM was investigated using a fluorescence resonance energy transfer assay. These binding experiments demonstrated that factor Va bound to vesicles containing 1, 5, 10, and 20% DOPS with Kd values of 2.88±0.43 nM, 1.15±0.17 nM, 1.16±0.13 nM, and 1.18±0.19 nM, respectively. At low concentrations of PS (≤ 5%), CH, DOPE and SM increased the binding affinity of factor Va 2 to 6-fold compared to vesicles containing only DOPS. When prothrombinase activity was measured as a function of factor Va concentration on vesicles containing 5, 10, and 20% DOPS, K1/2Va values of 0.63±0.08nM, 0.38±0.05nM, and 0.37±0.07nM were observed which were comparable to the Kd’s determined in direct factor Va binding experiments. These results indicate that factor Va binds tightly to phospholipid vesicles mimicking the phospholipid composition of activated platelets even at very low concentrations of PS. When direct binding of factor Xa to vesicles containing 5, 10, and 20% DOPS plus CH, DOPE and SM was examined, the Kd values were 70.5±6.8 nM, 62.6±6.6 nM, and 49.6±4.0 nM, respectively. When the interaction of factor Xa with membranes was investigated using prothrombinase assays, K1/2Xa values of 0.12±0.03nM, 0.04±0.01nM, and 0.01±0.01nM were observed. In conclusion, the presence of CH and PE increases the affinity of factor Va and factor Xa binding to phospholipid vesicles containing physiologic concentrations of PS. The binding of factor Va to vesicles containing 1% PS is not sufficient for assembly and function of the prothrombinase complex, suggesting that higher concentrations of PS are needed to support the binding of factor Xa and prothrombin.

Inhibition of Factor Xa in Prothrombinase Is Enhanced by Covalent Linkage of Antithrombin to Heparin.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1613-1613
Author(s):  
Sanjay Patel ◽  
Leslie R. Berry ◽  
Lesley Smith ◽  
Anthony Chan
Keyword(s):  
Rate Constants ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Complex Mixtures ◽  
Free State ◽  
Phospholipid Vesicles ◽  
Covalent Linkage ◽  
Rate Difference ◽  
Prothrombinase Complex ◽  
Factor Va

Abstract The rate of prothrombin to thrombin conversion by factor Xa (Xa) is enhanced when Xa is incorporated into the surface-bound prothrombinase complex. However, in comparison to the free state, Xa within the prothrombinase complex is afforded protection from antithrombin + heparin (AT+H) inactivation. We have shown that, unlike AT+H, a covalent conjugate of AT and H (ATH) can neutralize fibrin-bound thrombin. In this study, AT+H and ATH were compared in their reaction with Xa +/− prothrombinase complex. Mixtures of CaCl2, phospholipid vesicles, factor Va (Va) and prothrombin in TSP buffer, were combined with Xa. Following addition of either AT+H or ATH, time samples were neutralized with Na2EDTA + polybrene + substrate (S-2222) and residual Xa activity measured. Second order rate constants (k2) were calculated from plots of activity versus time. Results were compared to corresponding experiments with Xa alone. AT+H inactivation of Xa in prothrombinase occurred at a k2 (x 107 M−1min−1) of 2.34 +/− 0.09. In contrast, neutralization of free Xa by AT+H was significantly faster (k2 = 8.34 +/− 0.18, p = 0.03). Reaction with ATH showed no significant rate difference for Xa inhibition in either the complexed or free states (18.5 +/− 3.3 and 16.3 +/− 3.7, respectively). Intriguingly, the rates achieved for ATH inhibition of complexed and free Xa were significantly greater than that for AT+H with free Xa (p=0.03 and p=0.02, respectively). We conclude that covalent complexes of AT and H do not encounter resistance in the inhibition of Xa in prothrombinase, as seen for non-covalent AT+H mixtures. Thus, it is possible for ATH to effectively inhibit the propagation phase of thrombin generation and thus dampen thrombin production via neutralization of Xa in prothrombinase.

Regulation of Prothrombinase Activity by Protein S

1999 ◽  
Vol 82 (07) ◽  
pp. 80-87 ◽  
Author(s):  
Saulius Butenas ◽  
Neal Golden ◽  
Kenneth Mann ◽  
Cornelis van't Veer
Keyword(s):  
Tissue Factor ◽  
Protein C ◽  
Thrombin Generation ◽  
Plasma Concentrations ◽  
Factor Xa ◽  
Protein S ◽  
Coagulation Factors ◽  
Independent Effect ◽  
Factor Va ◽  
Prothrombinase Activity

SummaryThe independent effect of protein S as prothrombinase inhibitor has been proposed to depend on binding to both coagulation factors Va and factor Xa or on the binding to phospholipid thereby limiting the phospholipid available for prothrombinase activity. In this study we show that plasma concentrations of protein S (300 nM) equilibrated with the prothrombinase components (factor Va, factor Xa, phospholipid) cause a profound inhibition at low phospholipid concentrations (~0.2 μM). This inhibition by protein S of prothrombinase activity is abrogated with increasing phospholipid concentrations. Modeling of the effect of protein S on prothrombinase based only on the reported affinity of protein S for phospholipids (Kd ~ 10-8 M) in an equilibrium model (Clotspeed), predicted the experimentally obtained thrombin generation rates at low phospholipid in the presence of protein S based on the diminished available phospholipid binding sites for the prothrombinase components. Consistently, initial rates of prothrombinase activity are already maximally inhibited when protein S is preincubated with the phospholipid prior to the addition of factor Xa, factor Va and pro-thrombin. The results indicate that the order of addition of prothrombinase components and the availability of phospholipid may have a profound influence on observed effects of protein S on prothrombinase activity. All prothrombinase components (factor Xa, factor Va, phospholipid) become available during the course of the physiological thrombin generation. The effect of protein S was therefore studied on tissue factor-induced, platelet-dependent thrombin generation. Protein S delayed and inhibited the rate of thrombin generation of tissue factor-induced thrombin formation when surface is provided at physiologic concentrations using isolated platelets (2 × 108/ml). In contrast, protein S hardly affected thrombin generation in this model when platelets were pre-activated with collagen. Furthermore, the observed effects of addition of protein C and thrombomodulin in the absence or presence of protein S on tissue factor-induced, platelet-dependent thrombin generation, indicate that protein S and protein C may cooperate in the regulation of prothrombinase activity through independent mechanisms.

Endotoxin enhances the expression of monocyte prothrombinase activity

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 406-416 ◽  
Author(s):  
RA Robinson ◽  
L Worfolk ◽  
PB Tracy
Keyword(s):  
Mononuclear Cells ◽  
Membrane Surface ◽  
Factor Xa ◽  
Dependent Manner ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Flow Cytometric ◽  
And Function ◽  
Microscopy Techniques ◽  
Prothrombinase Activity

Abstract Thrombin is generated on the surface of mononuclear cells (MNCs) through the assembly and function of the prothrombinase complex consisting of the enzyme factor Xa, the cofactor/factor Va, calcium ions, and an appropriate membrane surface for proper assembly of the protein constituents. Assays performed in the presence of factors Va and Xa indicated that endotoxin significantly enhanced the prothrombinase activity (1.5- to 2.5-fold; P less than .001) expressed by MNCs in a dose- and time-dependent manner. Monocytes present in the MNC suspensions were responsible for this increased activity through processes resulting in both enhanced cellular activity and the enhanced release of membranous vesicles. Endotoxin was without effect on the expression of lymphocyte prothrombinase activity. Scanning electron microscopy techniques indicated that endotoxin resulted in extensive membrane blebbing of the monocytes present in the MNC suspensions with no effect on the morphology of the lymphocytes. Within 5 hours, endotoxin maximally enhanced the prothrombinase activity expressed by the monocyte membrane surface 2.8-fold, whereas 8 hours was required to maximally enhance the activity associated with the released vesicles by twofold. The observed increase in activity expressed by the monocyte membrane surface was due solely to endotoxin, since the activity expressed by the unstimulated monocyte membrane surface remained unaltered over time. In contrast, cell vesiculation, which occurred in the absence of any stimulus, was further enhanced by endotoxin. The increase in activity associated with the released vesicles from both stimulated and unstimulated cells paralleled an increase in the vesicle number as determined by flow cytometric analyses. The vesicle released from both unstimulated and stimulated monocytes were indistinguishable in size as determined by image analysis and ranged between 0.05 and 0.3 microns in diameter. 2-Deoxy-D-glucose (2DG) significantly enhanced the prothrombinase activity expressed by the monocyte membrane surface, as well as the released vesicle fraction, when used alone or in addition to endotoxin. The enhanced activity associated with the vesicle fraction again was attributed to the release of more vesicles. In contrast, cycloheximide decreased the prothrombinase activity expressed by the monocyte membrane surface, as well as the activity associated with vesicles released from both stimulated and unstimulated cells. These data suggest that the expression of monocyte prothrombinase activity can be significantly enhanced by endotoxin through processes that alter the monocyte membrane surface and augment the vesiculation process. Both processes appear to be regulated by protein synthesis and adenosine triphosphate (ATP)-dependent mechanisms.

scholarly journals Binding Site for Blood Coagulation Factor Xa Involving Residues 311–325 in Factor Va

1998 ◽  
Vol 273 (24) ◽  
pp. 14900-14905 ◽  
Author(s):  
Yumi Kojima ◽  
Mary J. Heeb ◽  
Andrew J. Gale ◽  
Tilman M. Hackeng ◽  
John H. Griffin
Keyword(s):  
Binding Site ◽  
Blood Coagulation ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Factor Va

The Role of Amino Acids 700–701 of the Factor Va Heavy Chain During Prothrombin Activation by Factor Xa

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2203-2203
Author(s):  
Jamila Hirbawi ◽  
Paul Y Kim ◽  
Michael E. Nesheim ◽  
Michael Kalafatis
Keyword(s):  
Amino Acids ◽  
Heavy Chain ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Human Origin ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Sds Page ◽  
Carboxyl Terminal ◽  
Prothrombin Activation

Abstract Abstract 2203 Blood coagulation is initiated after vascular injury, promoting formation of the fibrin plug. The prothrombinase complex plays a crucial role during activation of prothrombin (Pro) to thrombin. The complex is composed of the enzyme, factor Xa (fXa), along with its non-enzymatic cofactor, factor Va (fVa), in the presence of calcium on a phospholipid surface. The incorporation of fVa into the prothrombinase complex results in a 300,000-fold increase in the catalytic efficiency of fXa for thrombin generation. Prothrombinase activates prothrombin through initial cleavage at Arg320 followed by cleavage at Arg271 to yield human alpha-thrombin. This pathway is responsible for the generation of a transient catalytically active intermediate, meizothrombin. Recent data has suggested a differential effect of bovine and human factor Va on prothrombin-1 (Pre-1) activation by prothrombinase. This difference was localized within the last ten amino acids from the carboxyl-terminal region of fVa heavy chain. The only amino acid difference between the two cofactor molecules is localized at position 700–701 where the Asn-Arg dipeptide in the fVa of human origin is replaced by the Asp-Glu sequence in the carboxyl-terminal region of the cofactor of bovine origin. We have therefore constructed a recombinant human mutant fVa molecule with these amino acids mutated to their bovine counterpart. We have created a recombinant fVa molecule with the mutation700NR701 →DE. This recombinant cofactor molecule (fVDE) along with wild type factor V (fVWT) were transiently expressed in COS7 cells, purified to homogeneity, and assessed for their capability to by assembled in prothrombinase and promote Pro activation. Thrombin generation was evaluated by SDS-PAGE in a system using all proteins of human origin and the kinetic parameters of the reactions were determined using a chromogenic substrate to assess for thrombin activity. Kinetic analyses revealed that the Kd of fVaDE for human fXa, as well as the kcat and Km values of prothrombinase assembled with fVaDE for human Pro activation were similar to the values obtained following Pro activation by prothrombinase assembled with fVaWT. Surprisingly, SDS-PAGE analyses of prothrombin activation time courses revealed that the overall rate of cleavage of Pro by prothrombinase assembled with fVaDE was significantly delayed with significant accumulation of the intermediate meizothrombin, and delayed thrombin generation when compared to the rate of activation of Pro by prothrombinase assembled with fVaWT. Two-stage clotting assays (PT times) also revealed that fVaDE had reduced clotting activity when compared to fVaWT. Comparison of the rate of cleavage of two recombinant Pro mutant molecules, rMZ-II a recombinant Pro molecule that cannot be cleaved at Arg271 and rP2-II a recombinant Pro molecule that cannot be cleaved at Arg320, by prothrombinase assembled with fVaDE demonstrated impaired rate of cleavage of both substrates when compared to the rate of cleavage of the mutant recombinant Pro molecules by prothrombinase assembled with fVaWT. These findings were verified by experiments using active-site blocked purified human meizothrombin (FPR-meizo). Prothrombinase assembled with fVaDE was considerably impaired in its ability to cleave FPR-meizo at Arg271 as compared to the ability of prothrombinase assembled with fVaWT for the same cleavage. In fact, gel electrophoresis analyses demonstrated that prothrombinase assembled with fVaDE cleaves FPR-meizo with a rate similar to the cleavage of FPR-meizo by fXa alone. All these data together strongly suggest that the 700NR701 portion of the COOH-terminus of the fVa heavy chain plays a significant role in enzyme-substrate recognition/interaction during Pro activation by prothrombinase and thus regulates the rates of thrombin formation locally at the place of vascular injury. Disclosures: No relevant conflicts of interest to declare.

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