Roles of Platelets and Factor XI in the Initiation of Blood Coagulation by Thrombin

2001 ◽  
Vol 86 (07) ◽  
pp. 75-82 ◽  
Author(s):  
Peter Walsh
Keyword(s):  
Binding Site ◽  
Tissue Factor ◽  
Vascular Injury ◽  
Factor Xi ◽  
Platelet Surface ◽  
Proteolytic Activation ◽  
Activated Platelets ◽  
Tissue Factor Pathway ◽  
Protease Nexin ◽  
Normal Hemostasis

SummaryTo account for the variable hemostatic defect in patients with factor XI (FXI) deficiency, with normal hemostasis in contact factor deficiencies, a coagulation paradigm is presented whereby trace quantities of thrombin, generated transiently by exposure of tissue factor at sites of vascular injury, activates FXI bound to the platelet surface in the presence of prothrombin or high Mr kininogen (HK). Tissue factor pathway inhibitor (TFPI) limits the flux of thrombin generated by the tissue factor pathway, and protease nexin II (PNII), released from activated platelets, inhibits solution phase FXIa and localizes FIX activation to the platelet surface where FXIa is protected from inactivation by PNII. Either prothrombin or HK binds to the Apple 1 (A1) domain of FXI, thereby exposing a platelet-binding site in the FXI A3 domain. Dimeric FXI binds to activated platelets directly through the A3 domain of one monomer. After proteolytic activation of platelet-bound FXI by thrombin (or FXIIa), a substrate binding site for FIX is exposed in the opposite monomer that promotes FIX activation on the platelet surface resulting in the local explosive generation of thrombin and the formation of hemostatic thrombi at sites of vascular injury.

NMR Structural Analysis of Factor XI Apple 4 Domain.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1735-1735
Author(s):  
Dharmaraj Samuel ◽  
Hong Cheng ◽  
Paul W. Riley ◽  
Peter N. Walsh ◽  
Heinrich Roder
Keyword(s):  
Tissue Factor ◽  
Heavy Chain ◽  
Chemical Shifts ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Intrinsic Pathway ◽  
Factor Xi ◽  
Extrinsic Pathway ◽  
Platelet Surface ◽  
Normal Hemostasis

Abstract Blood coagulation is achieved by two closely coordinated mechanisms: i) the contact factor, or intrinsic, pathway initiated by assembly of coagulation proteins on negatively charged surfaces, and ii) the extrinsic pathway initiated by exposure of tissue factor at the site of vascular injury. Although factor XI (FXI), a 160 KDa homodimeric plasma coagulation protein, can be activated either by FXIIa (generated by the contact factor pathway) or by thrombin (generated by the extrinsic or tissue factor pathway), recent evidence suggests that its unique dimeric structure is required for FXI-activation by thrombin on the platelet surface, leading to the initiation of the intrinsic pathway that is required for normal hemostasis. Each FXI monomer consists of an N-terminal heavy chain and a C-terminal trypsin-like catalytic light chain. The heavy chain consists of four homologous subunits called apple domains (designated A1 to A4). The A2 and A3 domains of one monomeric subunit bind to FIX, whereas the A3 domain of the other monomeric subunit binds to platelets. The A4 domain, which shares high (25–38%) sequence identity with other Apple domains, facilitates FXI dimer formation through an intermolecular disulfide bond at Cys-321. In the present study the rA4 domain was cloned and purified to determine its three-dimensional structure. Multidimensional heteronuclear NMR experiments were carried out using C13, N15, H2 labeled samples. Chemical shifts of the C13, N15 and H1 resonance of all the residues were assigned. Assignment of NOE cross peaks between inter- and intra-subunit amino acids is in progress. Preliminary results indicate that the monomeric structure of the A4 domain consists of six anti-parallel β-strands and an α-helix, stabilized by three cystine cross links. The orientations of charged residues and hydrophobic patches on different sides of the molecule may play important roles in the dimerization process of FXI.

scholarly journals Activated factor XI increases the procoagulant activity of the extrinsic pathway by inactivating tissue factor pathway inhibitor

Blood ◽  
2015 ◽  
Vol 125 (9) ◽  
pp. 1488-1496 ◽  
Author(s):  
Cristina Puy ◽  
Erik I. Tucker ◽  
Anton Matafonov ◽  
Qiufang Cheng ◽  
Keith D. Zientek ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor X ◽  
Factor Xi ◽  
Extrinsic Pathway ◽  
Fibrin Formation ◽  
Key Points ◽  
Tissue Factor Pathway ◽  
Factor X Activation

Key Points Activated factor XI binds and proteolyzes tissue factor pathway inhibitor. Activated factor XI promotes factor X activation generation and fibrin formation through the inactivation of tissue factor pathway inhibitor from platelets and on endothelial cells.

A FVIII C1 Domain Loop Containing Phe2093 Contributes Affinity to the Binding of Recombinant FVIII C1C2 Proteins to Activated Platelets

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3069-3069
Author(s):  
Ting-Chang Hsu ◽  
Kathleen P. Pratt ◽  
Arthur R. Thompson
Keyword(s):  
Membrane Binding ◽  
Cysteine Residue ◽  
Wild Type ◽  
Platelet Surface ◽  
Proteolytic Activation ◽  
Mutant Proteins ◽  
Activated Platelets ◽  
C1 Domain ◽  
Beta Hairpin ◽  
The Difference

Abstract Factor VIII (FVIII) circulates bound to von Willebrand factor, and upon proteolytic activation it dissociates and attaches to activated membranes, e.g. at the platelet surface, that expose negatively-charged phosphatidylserine. This membrane association is mediated entirely by the FVIIIa light chain (A3-C1-C2), and the C2 domain is known to be a primary contributor to the membrane affinity. We previously demonstrated that the C1 domain, which is homologous to C2, also contributes to the affinity for activated platelets, (Hsu et al., Blood111, 200–208, 2008). Our earlier work showed that the platelet affinity of recombinant C1C2, as well as the total number of binding sites per platelet, were significantly higher than those measured for recombinant C2. Thus C1 either interacts with the platelet surface directly, or it stabilizes a conformation of C2 that promotes membrane binding. In this study, the affinities for activated platelets of a series of mutant C1C2 proteins were evaluated to determine residues involved in FVIII binding to platelets. C1C2 and C2 proteins were generated with a free cysteine residue substituted for the wild-type serine at position 2296 in C2 (S2296C does not disrupt the protein structure or affect membrane binding), to which a sulfyhydryl-linked fluorescein probe was attached covalently (C1C2* and C2*). Single or double alanine substitutions were introduced at the two beta-hairpin turns in C2 that mediate membrane binding (M2199A, F2200A, L2251A, L2252A), and at C1 residue F2093, which aligns with C2 residue L2252. Washed platelets were activated with SFLLRN-amide, incubated for 1 hr with the wild-type or mutant proteins, and analyzed by flow cytometry on a FACSCaliber. The relative binding affinities were estimated by using multiple protein concentrations and comparing the fluorescent signals to the values for saturation binding of C1C2* and C2*. Alanine substitutions at all of these positions resulted in decreased binding of C1C2*, comparable to the difference in affinity of C2* versus C1C2* (figure 1A). C1C2-F2093C was then generated, and the introduced sulfhydryl was blocked by adding free cysteine, thus introducing the bulky, charged cystine residue at this putative membrane-binding site (C1C2-F2093C-C). Binding assays utilizing detection by monoclonal antibody ESH8, which does not interfere with membrane attachment, followed by a PE-labeled secondary antibody showed that C1C2-F2093C-C had markedly reduced binding to activated platelets compared to C1C2 (figure 1B). These results are consistent with the hypothesis that the C1 domain contacts the membrane directly when FVIIIa becomes attached to the activated platelet surface and indicate that F2093 contributes significantly to this interaction. Figure Figure

scholarly journals Functional characterization of platelet-bound factor XIa: retention of factor XIa activity on the platelet surface

Blood ◽  
1986 ◽  
Vol 68 (1) ◽  
pp. 225-230 ◽  
Author(s):  
PN Walsh ◽  
D Sinha ◽  
A Koshy ◽  
FS Seaman ◽  
H Bradford
Keyword(s):  
Structural Integrity ◽  
Functional Characterization ◽  
Factor Ix ◽  
Scatchard Analysis ◽  
Factor Xi ◽  
Platelet Surface ◽  
Proteolytic Activation ◽  
Binding Data ◽  
Factor Xia ◽  
Activation Peptide

Previously we have shown that both factor XI and factor XIa are bound specifically to distinct, high-affinity sites on the surface of activated platelets in the presence of high Mr kininogen. To determine the functional significance of factor XIa binding to platelets, bound factor XIa has now been compared with the unbound enzyme. Platelets incubated with thrombin, high Mr kininogen, and 125I-labeled factor XIa bound 130 to 500 molecules of factor XIa per platelet. Scatchard analysis of binding data give a dissociation constant (Kd) of 822 pmol/L +/- 140 (SEM). Rates of factor IX activation, assayed by release of trichloroacetic acid-soluble 3H-labeled activation peptide from purified [3H]-factor IX, were similar when factor XIa was bound to platelets and when it was free in solution. The platelet-bound factor XIa was isolated by centrifugation through 20% sucrose and was functionally characterized both in a factor XIa coagulation assay and in the factor IX activation peptide release assay in comparison with unbound factor XIa in the presence of treated platelets. The functional activity of platelet-bound factor XIa as a factor IX activator as well as its structural integrity were shown to be fully retained on the platelet surface. Since platelets bind factor XI and promote its proteolytic activation to factor XIa, factor XIa binding to platelets may serve to localize factor IX activation to the hemostatic plug, where factor XIa is protected from inactivation by plasma protease inhibitors and where acceleration of subsequent coagulation reactions can occur.

Platelet Hyporeactivity in Very Low Birth Weight Neonates

1997 ◽  
Vol 77 (05) ◽  
pp. 1002-1007 ◽  
Author(s):  
Damodara Rajasekhar ◽  
Marc R Barnard ◽  
Francis J Bednarek ◽  
Alan D Michelson
Keyword(s):  
Flow Cytometry ◽  
Binding Site ◽  
Whole Blood ◽  
Very Low Birth Weight ◽  
Thromboxane A2 ◽  
Platelet Surface ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
Platelet Binding ◽  
Thromboxane A2 Analogue

SummaryVery few studies have examined platelet function in very low birth weight (VLBW) preterm neonates, because of the relatively large volumes of blood required. In this study, platelet function in clinically stable VLBW neonates was examined by whole blood flow cytometry, which requires only 5 |jl1 of whole blood per assay. The following monoclonal antibodies were used: S12 (P-selectin-specific, reflecting a granule secretion), PAC1 (directed against the fibrinogen binding site exposed on the GPIIb-IIIa complex of activated platelets), F26 (directed against a conformational change in fibrinogen bound to the GPIIb-IIIa complex), and 6D1 (directed against the von Willebrand factor binding site on the GPIb-IX-V complex). VLBW neonates, like normal adults, did not have circulating activated platelets, as determined by the lack of binding of SI2, PAC1, and F26 in the absence of an added agonist. VLBW neonatal platelets were markedly less reactive than adult platelets to thrombin, ADP/epinephrine, and U46619 (a stable thromboxane A2 analogue), as determined by the extent of increase in the platelet binding of SI2, PAC1, and F26, and the extent of decrease in the platelet binding of 6D1. In summary, compared to adults, the platelets of VLBW neonates are markedly hyporeactive to thrombin, ADP/epinephrine and a thromboxane A2 analogue in the physiologic milieu of whole blood, as determined by: 1) the increase in platelet surface P-selectin; 2) the exposure of the fibrinogen binding site on the GPIIb-IIIa complex; 3) fibrinogen binding; and 4) the decrease in platelet surface GPIb. This platelet hyporeactivity may be a factor in the propensity of VLBW neonates to intraventricular hemorrhage. In addition to its previously defined use as a test of platelet hyperreactivity, the present study suggests that whole blood flow cytometry may be useful in the clinical assessment of platelet hyporeactivity.

scholarly journals Functional characterization of platelet-bound factor XIa: retention of factor XIa activity on the platelet surface

Blood ◽  
1986 ◽  
Vol 68 (1) ◽  
pp. 225-230 ◽  
Author(s):  
PN Walsh ◽  
D Sinha ◽  
A Koshy ◽  
FS Seaman ◽  
H Bradford
Keyword(s):  
Structural Integrity ◽  
Functional Characterization ◽  
Factor Ix ◽  
Scatchard Analysis ◽  
Factor Xi ◽  
Platelet Surface ◽  
Proteolytic Activation ◽  
Binding Data ◽  
Factor Xia ◽  
Activation Peptide

Abstract Previously we have shown that both factor XI and factor XIa are bound specifically to distinct, high-affinity sites on the surface of activated platelets in the presence of high Mr kininogen. To determine the functional significance of factor XIa binding to platelets, bound factor XIa has now been compared with the unbound enzyme. Platelets incubated with thrombin, high Mr kininogen, and 125I-labeled factor XIa bound 130 to 500 molecules of factor XIa per platelet. Scatchard analysis of binding data give a dissociation constant (Kd) of 822 pmol/L +/- 140 (SEM). Rates of factor IX activation, assayed by release of trichloroacetic acid-soluble 3H-labeled activation peptide from purified [3H]-factor IX, were similar when factor XIa was bound to platelets and when it was free in solution. The platelet-bound factor XIa was isolated by centrifugation through 20% sucrose and was functionally characterized both in a factor XIa coagulation assay and in the factor IX activation peptide release assay in comparison with unbound factor XIa in the presence of treated platelets. The functional activity of platelet-bound factor XIa as a factor IX activator as well as its structural integrity were shown to be fully retained on the platelet surface. Since platelets bind factor XI and promote its proteolytic activation to factor XIa, factor XIa binding to platelets may serve to localize factor IX activation to the hemostatic plug, where factor XIa is protected from inactivation by plasma protease inhibitors and where acceleration of subsequent coagulation reactions can occur.

Factor XI Deficient Mice Have Reduced Platelet Accumulation and Fibrin Deposition after Laser Injury.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 218-218
Author(s):  
T. Regan Baird ◽  
David Gailani ◽  
Bruce Furie ◽  
Barbara C. Furie
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Thrombus Formation ◽  
Factor Xa ◽  
Fibrin Deposition ◽  
Wild Type ◽  
Factor Xi ◽  
Factor Xia ◽  
Tissue Factor Pathway ◽  
Platelet Accumulation

Abstract Tissue factor exposure at sites of vascular injury results in the generation of factor Xa and thrombin. A current model of blood coagulation suggests that the amount of thrombin generated through this pathway is limited by the inhibition of the factor VIIa-tissue factor complex by tissue factor pathway inhibitor in the presence of factor Xa. The initial thrombin activates a number of hemostatic proteins including factor XI. Factor XIa then activates factor IX leading to generation of the tenase complex to maintain the thrombin flux. While in vitro studies support this hypothesis the importance of factor XI for thrombus formation in vivo remains unclear. We have examined thrombus formation upon laser injury to the arterioles (30–50 μm diameter) of the cremaster muscle in living mice lacking factor XI using digital multi-channel fluorescence intravital microscopy. Platelets were labeled with Alexa 488 conjugated murine CD41 Fab fragments by systemic infusion of the antibody. Maximum platelet accumulation in factor XI null mice (median of 35 thrombi in 4 mice) is only 25% of that of wild type mice (median of 40 thrombi in 4 mice) after injury (p<0.03). The time course of platelet accumulation is similar between both genotypes. Maximum platelet accumulation occurs in approximately 90 seconds (p<0.2). Fibrin deposition was observed simultaneously using an Alexa 660 conjugated anti-fibrin antibody that does not recognize fibrinogen. Maximum fibrin deposition in factor XI null mice is 50% that of wild type mice (p<0.001) and the rate of fibrin generation is slower in factor XI null mice. However, the time to achieve half maximal fibrin deposition is approximately the same in factor XI null mice (77 sec) compared to wild type mice (63.5 sec, p<0.09). These data suggest that the primary difference in response to laser induced injury between the factor XI null mice and wild type mice is the level of thrombin generated and supports the hypothesis that factor XI participates in maintaining thrombin flux after inhibition of the factor VII-tissue factor. The model above postulates a single source of tissue factor, the vessel wall, and further, that the tissue factor-factor VIIa complex formed from the exposed tissue factor is rapidly inactivated by tissue factor pathway inhibitor after the appearance of the initial factor Xa formed. In addition it has been suggested that a rapidly growing thrombus blocks access to vascular wall tissue factor. However we have recently observed that there is a P-selectin and P-selectin glycoprotein ligand 1 dependent pathway of blood coagulation that recruits blood borne tissue factor into a growing thrombus at sites of laser-induced vessel injury. Both vessel wall and blood borne tissue factor are required for normal thrombus formation. Our data suggest that although tissue factor is continuously recruited to the growing thrombus, factor XIa plays a significant role in thrombin generation.

Regulation of tissue factor-induced coagulation and platelet aggregation in flowing whole blood

2005 ◽  
Vol 93 (01) ◽  
pp. 97-105 ◽  
Author(s):  
Marijke Kuijpers ◽  
Cécile Nieuwenhuys ◽  
Marion Feijge ◽  
Willem Kloots ◽  
Peter Giesen ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Whole Blood ◽  
Platelet Adhesion ◽  
Ex Vivo ◽  
Aggregate Formation ◽  
Ex Vivo Model ◽  
Platelet Surface ◽  
Inhibiting Effect ◽  
Activated Platelets ◽  
Adp Receptor

SummaryPhotochemically induced thrombosis (a thrombin-dependent process) was measured in rats treated with moderate doses of anticoagulants, but which appeared to be unchanged. We considered the possibility that platelet-inhibiting agents, which also indirectly inhibit coagulation, would act as more potent antithrombotic agents.Inhibitors used as such were prostaglandin E1 (PGE1), which elevates cyclic AMP levels, and the P2Y12 ADP-receptor antagonist,AR-C69931MX. Effects of these agents were investigated in an ex vivo model system, in which whole blood under coagulant conditions was perfused over fibrinogen at defined wall shear rate. Perfusion of blood (rat or human) in the presence of tissue factor resulted in deposition of activated platelets and subsequent aggregate formation, along with exposure of procoagulant phosphatidylserine (PS) on the platelet surface and formation of fibrin fibers. In the presence of PGE1 aggregation was completely inhibited,but platelet adhesion and PS exposure were only party reduced, while fibrin formation was hardly affected. Treatment with AR-C69931MX caused similar, but less complete effects.These results indicate that in tissue factor- triggered blood under conditions of flow:(i) the platelet procoagulant response is independent of aggregate formation; (ii) the platelet-inhibiting effect of PGE1 and AR-C69931MX is sufficient to suppress aggregation, but not platelet adhesion and coagulation. These platelet inhibitors thus maintain their aggregation- inhibiting effect at sites of thrombin formation.

scholarly journals Functional domains in the heavy-chain region of factor XI: a high molecular weight kininogen-binding site and a substrate-binding site for factor IX

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 244-251
Author(s):  
FA Baglia ◽  
D Sinha ◽  
PN Walsh
Keyword(s):  
Binding Site ◽  
Heavy Chain ◽  
Solid Phase ◽  
Competitive Inhibitor ◽  
Factor Ix ◽  
Functional Domains ◽  
Binding Studies ◽  
Factor Xi ◽  
Proteolytic Activation ◽  
Factor Xia

To probe the molecular interactions of factor XI we have prepared two monoclonal antibodies (MoAbs; 5F7 and 3C1), each of which binds the heavy chain of reduced and alkylated factor XIa. Competitive solid phase radioimmunoassay (RIA) binding studies revealed that 5F7 and 3C1 are directed against different epitopes within factor XI. One antibody (5F7) blocked the surface-mediated proteolytic activation of factor XI and its binding to HMW kininogen, but had no effect on factor-XIa- catalyzed factor IX activation. The other antibody (3C1) is a competitive inhibitor of factor-IX activation by factor XIa, but blocked factor-XI binding to HMW kininogen only at 1,000-fold higher concentration than 5F7. Moreover, HMW kininogen had no effect on the kinetics of factor-XIa-catalyzed factor-IX activation. Furthermore, factor XI CNBr peptide fragments that bind to the 5F7 and 3C1 antibodies were isolated. The peptides that bound to the 5F7 antibody blocked the binding of HMW kininogen to factor XI but did not inhibit factor-XIa-catalyzed factor-IX activation. However, the peptides isolated by the 3C1 antibody inhibited factor-XIa-catalyzed factor-IX activation and had no effect on factor-XI binding to HMW kininogen. Our results indicate that distinct functional domains within the heavy chain region of factor XI are important for the binding of factor XI to HMW kininogen and for activation of factor IX by factor XIa.

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