Lipid Raft Association of a Shared Factor X/Prothrombin Binding Site on Human Platelets Is Mediated by the Gla Domain.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 222-222
Author(s):  
Syed S. Ahmad ◽  
Peter N. Walsh
Keyword(s):  
Binding Site ◽  
Lipid Rafts ◽  
Structural Integrity ◽  
Gradient Centrifugation ◽  
Human Platelets ◽  
Factor X ◽  
High Affinity ◽  
Activated Platelets ◽  
Platelet Binding ◽  
20 Nm

Factor X (FX) initially binds to a high-capacity, low-affinity platelet binding site shared with prothrombin (FII) which then presents FX to a specific, high-affinity site consisting of FVIIIa bound to a high-affinity, low-capacity receptor on activated platelets. We have demonstrated the localization of FX in lipid rafts and shown that FX-raft association requires Ca2+ and is enhanced by saturating concentrations of FVIIIa. Here we investigate FII-raft association and define the domains through which shared FX and FII sites are mediated on the surface of human platelets. Activated (thrombin receptor peptide, SFLLRN, 25 μM) gel-filtered platelets (3.5 x 108/ml) were incubated with 125I-FII or 125I-FX to determine direct platelet binding and then they were lysed with Triton-X100 (0.025-0.25%) followed by sucrose density gradient centrifugation. FII was localized to lipid rafts in SFLLRN stimulated (~25% total binding) but not to unactivated platelets. The optimal associations of FX and FII with lipid rafts required Ca2+ and were not affected by the presence of EGR-FIXa or FIX (45 nM). The association of FII with lipid rafts was completely abolished in the presence of FX (1.5 μM) whereas, Gla (des) FX was unable to compete with raft associated FII. Similarly, 125I-FII fragment 1 association with lipid rafts was inhibited by FX but not by Gla (des) FX. Prothrombin and FII fragment 1 (residues 1-155) were equipotent inhibitors of FX-raft association. FVIIIa (20 nM) had no effect on FII-raft association but significantly increased (~2-fold to ~45%) FX-raft association. In contrast, the presence of FVa (20 nM) had no effect on FX-raft association but significantly (~2-fold to ~45%) increased FII-raft association. The structural integrity of lipid rafts was completely disrupted by 10 mM methyl-β-cyclodextrin (MβCD), a known cholesterol depleting drug, which completely prevented FII or FX association with lipid rafts, and this removal was reversed by cholesterol repletion. Furthermore, MβCD (up to 40 mM) had no effect on the amount of FII or FX bound to activated platelets, thus suggesting that neither platelet activation by SFLLRN nor the exposure of FII receptors was affected by MβCD treatment. These experiments demonstrate the localization of a shared FX/FII site in lipid rafts and support the hypothesis that these interactions are mediated by the Gla-domains of FX and FII and are specific and essential for the assembly of F-X activating complex on the activated platelet membrane.

A Binding Site Expressed on the Surface of Activated Human Platelets Is Shared by Factor X and Prothrombin†

Biochemistry ◽  
1996 ◽  
Vol 35 (27) ◽  
pp. 8890-8902 ◽  
Author(s):  
Joseph M. Scandura ◽  
Syed S. Ahmad ◽  
Peter N. Walsh
Keyword(s):  
Binding Site ◽  
Human Platelets ◽  
Factor X

The Role of Lipid Rafts in the Assembly and Down-Regulation of the Factor X Activating Complex on the Activated Platelet Membrane.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1010-1010 ◽  
Author(s):  
Syed S. Ahmad ◽  
Ya-Chi Su ◽  
Peter N. Walsh
Keyword(s):  
Lipid Rafts ◽  
Gradient Centrifugation ◽  
Early Time ◽  
Sucrose Density Gradient ◽  
Platelet Membrane ◽  
Membrane Microdomains ◽  
Factor X ◽  
Sucrose Density Gradient Centrifugation ◽  
Time Points ◽  
Substrate Complex

Abstract In a recent study of the role of detergent-insoluble platelet membrane microdomains (lipid rafts) in the assembly of the factor X (FX) activating complex, we have shown, contrary to expectations, that the formation of lipid rafts after incubation of platelets with the thrombin receptor activation peptide (SFLLRN, 25 μM, for 30 min) results in the down-regulation, rather than assembly, of the enzyme-cofactor-substrate complex by sequestering FVIIIa and FX in rafts and separating them from FIXa, which is excluded from raft fractions isolated by sucrose density gradient centrifugation of triton X-100 (0.25%) solubilized platelets. Since the FX-activating complex is assembled rapidly on platelets after incubation with low concentrations of either thrombin (>1 nM) or SFLLRN (25 μM), we have now examined the kinetics of FX-activation complex assembly at early time points after exposure of platelets to agonists. Washed and gel-filtered human platelets, activated with SFLLRN (25 μM) in the presence of FVIIIa (5 U/ml, 1.5 nM) and FX (125 nM) rapidly (within 2 min) developed the capacity to support maximal rates of FIXa (1 nM) catalyzed FX activation that was transient and decayed to baseline within 5 min after exposure to agonist. At these early time points (0.5, 1 and 2 min), platelets activated with SFLLRN (25 μM) in the presence of 125I-labled FVIIIa (nM), FIXa (nM) or FX (nM) and analyzed by sucrose density gradient centrifugation after solubilization in triton X-100 (0.25%) were shown to sequester within the raft fractions ~15% of FIXa, ~15% of FVIIIa and ~15% of FX, whereas at later time points (5–30 min) only FVIIIa (~25%) and FX (~45%) were localized in rafts, from which FIXa was completely excluded. These results strongly suggest that platelet membrane microdomains (lipid rafts) form rapidly after exposure of platelets to PAR-1 agonists to colocalize the enzyme-cofactor-substrate complex, which is transient since at later time points FIXa dissociates from rafts that sequester the FVIIIa-FX complex to down-regulate FX activation.

scholarly journals The effect of thrombin on the density distribution of blood platelets: detection of activated platelets in the circulation

Blood ◽  
1983 ◽  
Vol 62 (2) ◽  
pp. 433-438
Author(s):  
B van Oost ◽  
IH van Hien-Hagg ◽  
AP Timmermans ◽  
JJ Sixma
Keyword(s):  
Cardiopulmonary Bypass ◽  
Density Distribution ◽  
Density Gradient Centrifugation ◽  
Blood Platelets ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Human Platelets ◽  
Activated Platelets

The buoyant density of human platelets is decreased after they have been aggregated and induced to secrete their granule content by thrombin. This change in density was detected by discontinuous density gradient centrifugation using arabinogalactan (Stractan) solutions. The density decrease was dependent on the thrombin concentration and paralleled the extent of serotonin and beta-thromboglobulin secretion. The degranulated platelets maintained their integrity, and many of their functional properties. Mixtures of degranulated platelets and normal platelets could be resolved by Stractan gradient centrifugation and the number of degranulated platelets quantitated. Using this method, increased levels of less dense platelets were shown to occur after cardiopulmonary bypass. Assay of changes in platelet density by Stractan gradient centrifugation is a useful method for detection of activated platelets in vitro and in vivo.

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.

The Effects Of Chymotrypsin And Of ADP On The Binding Site For Bovine Factor VIII On Human Platelets

1981 ◽  
Author(s):  
Edward P Kirby ◽  
David C B Mills
Keyword(s):  
Factor Viii ◽  
Binding Site ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Shape Change ◽  
Human Platelets ◽  
Simple Process ◽  
Affecting Factor ◽  
Platelet Binding ◽  
Bovine Factor

The aggregation of human platelets by bovine Factor VIII (Platelet Agglutinating Factor-PAF) is inhibited by exposure of the cells to ADP or Chymotrypsin. We have investigated the mechanism of these effects using washed platelets. The washing procedure was modified from the method of Mustard et al. (Brit. J. Haematol. 22:193, 1972), omitting heparin and using a protein-free Tyrode’s solution for the final resuspension. The washed platelets were stable and responded to ADP (0.1-1 μM) with a shape change and, if fibrinogen was added, with aggregation. Bovine Factor VIII was purified to >90% homogeneity and was labeled with 125I (approx. 1 atom/subunit) by the IodoGen procedure, with no loss of activity. Aggregation was measured in the aggregometer in the presence of 7 mM EDTA. Binding was measured after incubation of labeled Factor VIII with washed platelets in the presence of 7 mM EDTA for 5 min at 37° without stirring.Treatment of washed platelets with Chymotrypsin progressively destroyed their ability to bind Factor VIII and to be agglutinated by it. Responsiveness to Factor VIII disappeared before any alteration was detected in the ability of platelets to undergo ADP-induced shape change. Treatment of platelets with ADP, however, inhibited agglutination induced by Factor VIII without affecting the binding of Factor VIII to the platelets. Agglutination by wheat germ agglutinin or phytohemagglutinin was not inhibited by ADP treatment. We conclude that Chymotrypsin probably inhibits Factor VIII- induced agglutination by destroying the platelet binding site for Factor VIII, but that ADP must act at a point distal to Factor VIII binding. Agglutination of metabolically intact platelets by Factor VIII may not be a simple process, because ADP can specifically inhibit it without affecting Factor VIII binding or aggregation of the platelets by lectins.

Alteration of the Factor X Zymogen to Protease Transition Provides Evidence for Allosteric Linkage between the S1 and FVa Binding Sites.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 30-30 ◽  
Author(s):  
Raffaella Toso ◽  
Hua Zhu ◽  
Rodney M. Camire
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Membrane Surface ◽  
Coagulation Factor ◽  
Salt Bridge ◽  
Kinetic Studies ◽  
Factor X ◽  
Bridge Formation ◽  
High Affinity ◽  
Km Value

Abstract The zymogen to protease transition in the chymotrypsin-like serine protease family follows a well described mechanism in which bond cleavage at a highly conserved site (Arg15-Ile16; chymotrypsin numbering system) results in the unmasking of a new N-terminus that acts as an intramolecular ligand for Asp194. This new salt-bridge drives a conformational change in the so-called “activation domain”, surface loops consisting of the S1 specificity pocket, oxyanion hole, autolysis loop, and sodium biding site. It is well documented in the trypsin system that Ile16-Asp194 internal salt-bridge formation is allosterically linked to the S1 specificity site; that is changes at one site influence the other and vice versa. Blood coagulation factor Xa (FXa) reversibly associates with its cofactor factor Va (FVa) on a membrane surface in the presence of Ca2+ ions with high affinity; an interaction which is not mimicked by the zymogen FX. To determine whether the FX zymogen to protease transition contributes to the expression of a high affinity FVa binding site, we constructed a series of FXa variants which are shifted along this transition pathway. To generate these “zymogen-like” proteins, we made several substitutions at position 16 or 17, with the intent of destabilizing the intramolecular salt bridge to varying degrees. Following a series of preliminary experiments, three mutants were chosen for expression, purification, and activation with RVV-X: I16L, I16G, and V17A. Kinetic studies using peptidyl substrates and active site directed probes revealed that I16L and V17A have an impaired ability to bind these probes (15 to 25-fold increase in the Km or Ki) while the rate of catalysis (kcat) was reduced by 3-fold compared to wild-type FXa (wtFXa; plasma-derived and recombinant). The I16G variant was not inhibited by any of the probes examined and its chromogenic activity was severely impaired (>500 to 1000-fold), precluding calculation of kinetic parameters. These data are consistent with the idea that destabilization of internal salt-bridge formation (Ile16-Asp194) influences binding at the S1 specificity site. In contrast to these results, assembly of I16L and V17A into prothrombinase almost completely restored the Km for peptidyl substrates while the kcat was still 3-fold reduced, indicating that FVa binding can rescue binding at the active site. Surprisingly, even the Km value for I16G was almost completely restored (3-fold increased compared to wtFXa) when assembled in prothrombinase; however a 60-fold reduction in the kcat was found. Consistent with these data, kinetic studies using prothrombin or prethrombin-1 revealed that each of the FXa variants had a normal Km value when assembled in prothrombinase; while the kcat values where reduced to a similar extent as for the chromogenic substrates. Overall our data indicate that direct binding of these FXa variants to FVa rescues binding at S1 site, suggesting allosteric linkage exists between these sites. Thus the FX zymogen to protease transition not only influences the formation of the S1 pocket, but also contributes in a substantial way to the formation of a FVa binding site.

Endothelial Protein C Receptor Is Expressed on Procoagulant Platelets and Contributes to Factor VIIa Binding and Activity

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4224-4224 ◽  
Author(s):  
Ammon M. Fager ◽  
Maureane Hoffman
Keyword(s):  
Endothelial Cell ◽  
Research Funding ◽  
Protein C ◽  
Thrombin Generation ◽  
Factor Viia ◽  
Human Platelets ◽  
Activated Platelets ◽  
Platelet Binding ◽  
Hemostatic Efficacy ◽  
Maximal Binding

Abstract Recombinant factor VIIa (rFVIIa) is routinely used as an effective bypassing agent to promote hemostasis in hemophilia patients with inhibitory antibodies that compromise factor replacement. In addition, rFVIIa is extensively used off-label as a hemostatic agent in cardiovascular surgery, trauma, and intracranial hemorrhage. Attempts to improve the treatment for these patients have included the production of rFVIIa analogs such as V158D/E296V/M298Q-FVIIa (FVIIa-DVQ). Previous studies have shown that FVIIa-DVQ exhibits enhanced in vitro procoagulant and antifibrinolytic activity, greater factor Xa (FXa) and thrombin generation on activated platelets, and improved hemostatic efficacy relative to wild-type FVIIa in a mouse model of hemophilia. Surprisingly, while FVIIa-DVQ and rFVIIa bind similarly to phospholipid vesicles, FVIIa-DVQ exhibits greater binding to platelets than rFVIIa. It has been established that only a fraction of activated platelets are able to bind high levels of coagulation factors. These highly procoagulant platelets, often called COATed platelets, also preferentially bind rFVIIa and FVIIa-DVQ in a Gla-domain dependent manner. However, the exact mechanism of this interaction remains largely unknown. As previous studies have shown that the endothelial cell protein C receptor (EPCR) also functions as the endothelial cell receptor for FVIIa, the purpose of the current study was to determine whether an interaction with EPCR might be responsible for the increased platelet binding and hemostatic efficacy of FVIIa-DVQ. Following their isolation from anticoagulated whole blood, human platelets were activated with a combination of thrombin plus the collagen receptor agonist convulxin, in order to generate highly procoagulant platelets. We then examined the binding of both rFVIIa and FVIIa-DVQ by flow cytometry in the presence and absence of excess protein C (PC) to determine its ability to compete for platelet binding. As previously reported, maximal binding of FVIIa-DVQ was significantly higher than the maximal binding of rFVIIa in the absence of PC. Interestingly, the addition of PC inhibited the binding of FVIIa-DVQ to a greater extent than the binding of rFVIIa, thereby eliminating the difference in binding seen in the absence of PC. Using both FXa and thrombin generation assays we found that this competition for platelet binding resulted in a corresponding decrease in the procoagulant activity of FVIIa-DVQ. Similar experiments were also performed to evaluate rFVIIa and FVIIa-DVQ binding to platelets in the presence and absence of a rabbit anti-EPCR antibody. Equivalent decreases in the platelet binding and activity of both molecules were seen in the presence of this antibody, thereby confirming that these results are indeed due to interactions with EPCR. However, while mRNA encoding EPCR has been reported in genome-wide sequencing of the human platelet transcriptome, EPCR protein expression has not previously been shown in platelets. We therefore conducted flow cytometric analyses of unactivated, thrombin activated, and thrombin plus convulxin activated platelets to evaluate their expression of EPCR. We found that EPCR is not expressed on either unactivated or thrombin-activated platelets. Conversely, the highly procoagulant platelets do express EPCR. Dual-labeling studies confirmed that those platelets which express EPCR also bind the most rFVIIa. To confirm platelet EPCR expression we first confirmed the presence of EPCR pre-mRNA in unactivated platelets by RT-PCR. We then determined the presence of EPCR protein by immunoprecipitation from unactivated platelet lysates followed by western blotting and mass spectrometric analyses. These data unambiguously demonstrate that EPCR is present in unactivated platelets, and is specifically expressed by the highly procoagulant platelet subpopulation. This work represents the first demonstration that human platelets are capable of expressing EPCR, and suggests that EPCR plays a role in the efficacy of rFVIIa as a therapeutic agent by contributing to platelet-FVIIa interactions. A better understanding of the mechanism by which rFVIIa binds to the activated platelet will facilitate the development of new therapeutic agents to improve the treatment and quality of life for patients requiring emergency hemostasis. Disclosures Hoffman: CSL-Behring: Consultancy, Research Funding; Boehringer Ingelheim: Research Funding; Novo Nordisk: Honoraria, Research Funding.

scholarly journals The effect of thrombin on the density distribution of blood platelets: detection of activated platelets in the circulation

Blood ◽  
1983 ◽  
Vol 62 (2) ◽  
pp. 433-438 ◽  
Author(s):  
B van Oost ◽  
IH van Hien-Hagg ◽  
AP Timmermans ◽  
JJ Sixma
Keyword(s):  
Cardiopulmonary Bypass ◽  
Density Distribution ◽  
Density Gradient Centrifugation ◽  
Blood Platelets ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Human Platelets ◽  
Activated Platelets

Abstract The buoyant density of human platelets is decreased after they have been aggregated and induced to secrete their granule content by thrombin. This change in density was detected by discontinuous density gradient centrifugation using arabinogalactan (Stractan) solutions. The density decrease was dependent on the thrombin concentration and paralleled the extent of serotonin and beta-thromboglobulin secretion. The degranulated platelets maintained their integrity, and many of their functional properties. Mixtures of degranulated platelets and normal platelets could be resolved by Stractan gradient centrifugation and the number of degranulated platelets quantitated. Using this method, increased levels of less dense platelets were shown to occur after cardiopulmonary bypass. Assay of changes in platelet density by Stractan gradient centrifugation is a useful method for detection of activated platelets in vitro and in vivo.

scholarly journals Characterization of the gamma chain platelet binding site on fibrinogen fragment D

Blood ◽  
1992 ◽  
Vol 79 (10) ◽  
pp. 2643-2648 ◽  
Author(s):  
NE Kirschbaum ◽  
MW Mosesson ◽  
DL Amrani
Keyword(s):  
Platelet Aggregation ◽  
Binding Site ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Gamma Chain ◽  
Platelet Surface ◽  
Fibrinogen Binding ◽  
Platelet Binding ◽  
Chain Sequence ◽  
Carboxy Terminal

Abstract Glycoprotein (GP) IIb/IIIa on adenosine diphosphate (ADP)-activated human platelets interacts with specific sites on the fibrinogen molecule leading to aggregation. We characterized the platelet-binding site on the gamma chains of fibrinogen using plasmic fragments D gamma A and D gamma'. Fragment D gamma A, which contains the carboxy terminal gamma A400–411 platelet-binding sequence (HHLGGAKQAGDV), was 70-fold more active than the synthetic gamma A400–411 peptide in inhibiting ADP- induced platelet aggregation. Fragment D gamma A inhibited fibrinogen binding and also bound directly to ADP-activated platelets. The Kd values determined for fibrinogen and fragment D gamma A binding were 0.55 mumol/L and 1.2 mumol/L, respectively. In contrast, fragment D gamma', which differs from fragment D gamma A with respect to its gamma chain sequence from position 408 to the COOH-terminus at position 427, did not inhibit platelet aggregation or fibrinogen binding, and did not bind directly to the platelet surface. Denaturation of fragment D gamma A with guanidine-HCl caused a loss of inhibitory activity in platelet aggregation assays. These data indicate that the native conformation of the gamma chain platelet-binding site on fibrinogen is important for optimal binding to GPIIb/IIIa.

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