Complement proteins C5b-9 stimulate procoagulant activity through platelet prothrombinase

Abstract The capacity of platelets treated with nonlytic concentrations of the C5b-9 proteins to catalyze prothrombin activation and thereby trigger clot formation has been investigated. When suspended in the presence of exogenous factors Xa and Va, gel-filtered platelets treated with purified C5b-9 proteins catalyzed prothrombin to thrombin conversion at rates up to tenfold above controls, and exceeded by up to fourfold the prothrombinase activity observed for thrombin-stimulated platelets. In the absence of added factor Va, C5b-9 assembly on the platelet surface significantly shortened the lag period before prothrombinase expression that was observed for untreated platelets and increased the maximum catalytic rate of thrombin formation. A comparison with other platelet stimuli revealed that the C5b-9-induced activation of platelet prothrombinase closely paralleled the effects mediated by calcium ionophore A23187. Our data suggest that the C5b-9 proteins promote the release of platelet factor V and the assembly of the prothrombinase complex, thereby potentiating the effects of thrombin on the activation of prothrombinase. Membrane assembly of the C5b-9 proteins was also observed to markedly accelerate the rate of platelet-catalyzed plasma clotting, suggesting a direct link between C5b-9-mediated prothrombinase activation and procoagulant activity accompanying immunologic damage to the platelet.

Download Full-text

Complement proteins C5b-9 stimulate procoagulant activity through platelet prothrombinase

Blood ◽

10.1182/blood.v68.4.875.bloodjournal684875 ◽

1986 ◽

Vol 68 (4) ◽

pp. 875-880 ◽

Cited By ~ 3

Author(s):

T Wiedmer ◽

CT Esmon ◽

PJ Sims

Keyword(s):

Calcium Ionophore ◽

Calcium Ionophore A23187 ◽

Procoagulant Activity ◽

Ionophore A23187 ◽

Factor V ◽

Platelet Factor ◽

Platelet Surface ◽

Prothrombinase Complex ◽

Factor Va ◽

Exogenous Factors

The capacity of platelets treated with nonlytic concentrations of the C5b-9 proteins to catalyze prothrombin activation and thereby trigger clot formation has been investigated. When suspended in the presence of exogenous factors Xa and Va, gel-filtered platelets treated with purified C5b-9 proteins catalyzed prothrombin to thrombin conversion at rates up to tenfold above controls, and exceeded by up to fourfold the prothrombinase activity observed for thrombin-stimulated platelets. In the absence of added factor Va, C5b-9 assembly on the platelet surface significantly shortened the lag period before prothrombinase expression that was observed for untreated platelets and increased the maximum catalytic rate of thrombin formation. A comparison with other platelet stimuli revealed that the C5b-9-induced activation of platelet prothrombinase closely paralleled the effects mediated by calcium ionophore A23187. Our data suggest that the C5b-9 proteins promote the release of platelet factor V and the assembly of the prothrombinase complex, thereby potentiating the effects of thrombin on the activation of prothrombinase. Membrane assembly of the C5b-9 proteins was also observed to markedly accelerate the rate of platelet-catalyzed plasma clotting, suggesting a direct link between C5b-9-mediated prothrombinase activation and procoagulant activity accompanying immunologic damage to the platelet.

Download Full-text

Defective Ca(2+)-induced microvesiculation and deficient expression of procoagulant activity in erythrocytes from a patient with a bleeding disorder: a study of the red blood cells of Scott syndrome

Blood ◽

10.1182/blood.v79.2.380.bloodjournal792380 ◽

1992 ◽

Vol 79 (2) ◽

pp. 380-388 ◽

Cited By ~ 1

Author(s):

EM Bevers ◽

T Wiedmer ◽

P Comfurius ◽

SJ Shattil ◽

HJ Weiss ◽

...

Keyword(s):

Red Blood Cells ◽

Blood Cells ◽

Calcium Ionophore ◽

Coagulation Factors ◽

Calcium Ionophore A23187 ◽

Bleeding Disorder ◽

Procoagulant Activity ◽

Ionophore A23187 ◽

Factor Va ◽

Resealed Ghosts

The erythrocytes from a patient with Scott syndrome, a bleeding disorder characterized by an isolated defect in expression of platelet procoagulant activity, have been studied. When incubated with the calcium ionophore A23187, Scott syndrome red blood cells (RBCs) expressed less than 10% of the prothrombinase (enzyme complex of coagulation factors Va and Xa) activity of A23187-treated RBCs obtained from normal controls. Consistent with the results from enzyme assay, the ionophore-treated Scott syndrome erythrocytes exhibited diminished membrane vesiculation and decreased exposure of membrane binding sites for factor Va compared with identically treated controls. When examined by scanning electron microscopy, untreated Scott syndrome RBCs were indistinguishable from normal cells. After incubation with A23187, however, the morphology of Scott syndrome RBCs contrasted markedly from normal erythrocytes. Whereas the Ca2+ ionophore induced marked echinocytosis and spiculation of normal RBCs, Scott syndrome RBCs remained mostly discoid under these conditions, with only an occasional echinocyte-like cell observed. These aberrant responses to intracellular Ca2+ were also observed for resealed ghosts prepared from Scott syndrome erythrocytes, indicating that they are related to a defect in the membrane or membrane-associated cytoskeleton. The finding that the erythrocytes of this patient share many of the membrane abnormalities reported previously for Scott syndrome platelets suggests that this defect is common to both cell lines and involves a membrane component required for vesicle formation and for expression of prothrombinase sites.

Download Full-text

Defective Ca(2+)-induced microvesiculation and deficient expression of procoagulant activity in erythrocytes from a patient with a bleeding disorder: a study of the red blood cells of Scott syndrome

Blood ◽

10.1182/blood.v79.2.380.380 ◽

1992 ◽

Vol 79 (2) ◽

pp. 380-388 ◽

Cited By ~ 94

Author(s):

EM Bevers ◽

T Wiedmer ◽

P Comfurius ◽

SJ Shattil ◽

HJ Weiss ◽

...

Keyword(s):

Red Blood Cells ◽

Blood Cells ◽

Calcium Ionophore ◽

Coagulation Factors ◽

Calcium Ionophore A23187 ◽

Bleeding Disorder ◽

Procoagulant Activity ◽

Ionophore A23187 ◽

Factor Va ◽

Resealed Ghosts

Abstract The erythrocytes from a patient with Scott syndrome, a bleeding disorder characterized by an isolated defect in expression of platelet procoagulant activity, have been studied. When incubated with the calcium ionophore A23187, Scott syndrome red blood cells (RBCs) expressed less than 10% of the prothrombinase (enzyme complex of coagulation factors Va and Xa) activity of A23187-treated RBCs obtained from normal controls. Consistent with the results from enzyme assay, the ionophore-treated Scott syndrome erythrocytes exhibited diminished membrane vesiculation and decreased exposure of membrane binding sites for factor Va compared with identically treated controls. When examined by scanning electron microscopy, untreated Scott syndrome RBCs were indistinguishable from normal cells. After incubation with A23187, however, the morphology of Scott syndrome RBCs contrasted markedly from normal erythrocytes. Whereas the Ca2+ ionophore induced marked echinocytosis and spiculation of normal RBCs, Scott syndrome RBCs remained mostly discoid under these conditions, with only an occasional echinocyte-like cell observed. These aberrant responses to intracellular Ca2+ were also observed for resealed ghosts prepared from Scott syndrome erythrocytes, indicating that they are related to a defect in the membrane or membrane-associated cytoskeleton. The finding that the erythrocytes of this patient share many of the membrane abnormalities reported previously for Scott syndrome platelets suggests that this defect is common to both cell lines and involves a membrane component required for vesicle formation and for expression of prothrombinase sites.

Download Full-text

Increased expression of procoagulant activity on the surface of human platelets exposed to heavy-metal compounds

Biochemical Journal ◽

10.1042/bj3080015 ◽

1995 ◽

Vol 308 (1) ◽

pp. 15-21 ◽

Cited By ~ 6

Author(s):

C A Goodwin ◽

C P D Wheeler-Jones ◽

S Namiranian ◽

S Bokkala ◽

V V Kakkar ◽

...

Keyword(s):

Heavy Metal ◽

Calcium Ionophore ◽

Calcium Ionophore A23187 ◽

Procoagulant Activity ◽

Ionophore A23187 ◽

Dependent Manner ◽

Anionic Phospholipid ◽

Metal Compounds ◽

Platelet Surface ◽

Thrombin Formation

One of the essential roles for platelets in haemostasis is in the potentiation of blood clotting due to the contribution of anionic phospholipid from the surface of the cells, as an essential cofactor to the proteolytic reactions of coagulation (platelet procoagulant activity). Only a limited number of agonists are known to initiate platelet procoagulant activity. In this study the rate of thrombin formation on the platelet surface was observed to increase in a dose-dependent manner upon treatment of washed platelets with heavy-metal compounds. Unlike the immediate increase observed upon treatment of platelets with calcium ionophore, A23187, the change due to these agents was progressive, approaching a maximum after 10 min. The maximum-fold acceleration of the rate of thrombin formation compared with control platelets was calculated for HgCl2 (56-fold), AgNO3 (42-fold) phenylmercuriacetate (24-fold) and thimerosal (14-fold), compared with 70-fold observed for calcium ionophore. The increase in procoagulant activity due to HgCl2 coincided with a large increase in intracellular calcium and phosphorylation of 22 and 45 kDa proteins. It is considered that the mechanism responsible for the increase in procoagulant activity is exposure of anionic phospholipids. This was detected by a 2-fold increase in the binding of 125I-annexin V upon addition of HgCl2, compared with resting platelets (3-fold on treatment of platelets with calcium ionophore). In contrast to the generation of activity by A23187 and other known agonists of this reaction, heavy-metal compounds appeared to cause little or no release of microparticles from the platelet surface. Since HgCl2 did not cause aggregation of platelets or significant release of serotinin, these findings may give further support to the need for exposure and ligation of glycoprotein IIb:IIIa for vesiculization to occur. Treatment of platelets with heavy metals may constitute a new approach to investigating the early changes in the cell membrane which lead to increased expression of anionic phospholipid.

Download Full-text

Activation/inactivation of human factor V by plasmin

Blood ◽

10.1182/blood.v73.1.185.185 ◽

1989 ◽

Vol 73 (1) ◽

pp. 185-190 ◽

Cited By ~ 100

Author(s):

CD Lee ◽

KG Mann

Keyword(s):

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Coagulation Factor ◽

Temporal Correlation ◽

Procoagulant Activity ◽

Factor V ◽

Prothrombinase Complex ◽

Factor Va ◽

Active Fragments ◽

Transient Intermediates

Abstract The effect of human plasmin on human coagulation factor V was studied using isolated proteins. Incubation of factor V with plasmin resulted in a rapid increase in procoagulant activity, followed by a subsequent decline in the ability of factor V to serve as a cofactor in the prothrombinase complex. Identical results were obtained when these reactions were conducted in the presence of dansylarginine-N-(3-ethyl- 1,5-pentanediyl) amide (DAPA), indicating that the changes observed could not have occurred as a consequence of cleavage by alpha-thrombin. Analysis of the products of the reaction by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) revealed a temporal correlation between the rise and fall in factor V activity and the presence of several transient intermediates. These fragments are distinct from the subunits of alpha-thrombin-activated factor V (factor Va). The activation phase of the reaction was not significantly affected by the presence of phospholipid. In contrast, the rate of degradation of active fragments of factor V and the accompanying loss of activity were markedly enhanced in the presence of phospholipid vesicles. These data suggest that the action of plasmin upon factor V results in the transient formation of proteolytic fragments which express significant procoagulant activity.

Download Full-text

Platelet microparticle membranes have 50- to 100-fold higher specific procoagulant activity than activated platelets

Thrombosis and Haemostasis ◽

10.1160/th06-06-0313 ◽

2007 ◽

Vol 97 (03) ◽

pp. 425-434 ◽

Cited By ~ 283

Author(s):

Dmitry Kireev ◽

Nadezhda Popenko ◽

Aleksei Pichugin ◽

Mikhail Panteleev ◽

Olga Krymskaya ◽

...

Keyword(s):

Blood Platelets ◽

Calcium Ionophore ◽

Coagulation Factors ◽

In Vitro Models ◽

Calcium Ionophore A23187 ◽

Procoagulant Activity ◽

Ionophore A23187 ◽

Factor X ◽

Activated Platelets

SummaryPlatelet microparticles (PMPs) are small vesicles released from blood platelets upon activation. The procoagulant activity of PMPs has been previously mainly characterized by theirability to bind coagulation factors VIII and Va in reconstructed systems. It can be supposed that PMPs can contribute to the development of thrombotic complications in the pathologic states associated with the increase of their blood concentration. In this study we compared procoagulant properties of calcium ionophore A23187-activated platelets and PMPs using several in-vitro models of hemostasis. Surface densities of phosphatidylserine, CD61, CD62P and factor X bound per surface area unit were determined by flow cytometry. They were 2.7-, 8.4-, 4.3-, and 13-fold higher for PMPs than for activated platelets, respectively. Spatial clot growth rate (Vclot) in the reaction-diffus ion experimental model and endogenous thrombin potential (ETP) were determined in plasma, which was depleted of phospholipid cell surfaces by ultra-centrifugation and supplemented with activated platelets or PMPs at different concentrations. Both Vcllot and ETP rapidly increased with the increase of PMP or platelet concentration until saturation was reached. The plateau values of Vclot and ETP for activated platelets and PMPs were similar. In both assays, the procoagulant activity of one PMP was almost equal to that of one activated platelet despite at least two-orders-of-magnitude difference in their surface areas. This suggests that the PMP surface is approximately 50- to 100-fold more procoagulant than the surface of activated platelets.

Download Full-text

The effect of platelets upon factor Xa-catalyzed activation of factor VII in vitro

Blood ◽

10.1182/blood.v72.2.396.396 ◽

1988 ◽

Vol 72 (2) ◽

pp. 396-401 ◽

Cited By ~ 6

Author(s):

LV Rao ◽

SI Rapaport

Keyword(s):

Surface Membrane ◽

Calcium Ionophore ◽

Factor Xa ◽

Factor Vii ◽

Ionophore A23187 ◽

Exogenous Factor ◽

Platelet Factor ◽

Anionic Phospholipids ◽

Factor Va ◽

Activated Platelets

Abstract The authors have investigated the ability of platelets to enhance factor Xa-catalyzed activation of factor VII. Unstimulated platelets were without effect, whereas freeze/thawed platelets substantially enhanced activation. Antifactor V antibodies did not diminish the enhancement. Platelets activated by thrombin, collagen, or calcium ionophore A23187 also enhanced factor Xa-catalyzed activation of factor VII. In contrast to their lack of effect upon freeze/thawed platelets, antifactor V antibodies abolished augmented factor VII activation induced by activated platelets. Adding exogenous factor Va to unstimulated platelets failed to enhance factor Xa-catalyzed activation of factor VII, nor did adding exogenous factor Va to activated platelets augment activation beyond that observed with activated platelets alone. These observations can be interpreted as follows: (1) factor Va does not function as a cofactor for factor Xa-catalyzed activation of factor VII; (2) anionic phospholipids are a known cofactor for factor Xa-catalyzed activation of factor VII, and freeze/thawed platelets probably enhance activation by making anionic phospholipids on disrupted platelet membranes available to function as a cofactor; (3) the presumed binding of factor Xa to exogenous factor Va on unstimulated platelets is insufficient in itself to augment factor Xa-catalyzed activation of factor VII; (4) activated platelets augment factor Xa-catalyzed factor VII activation because activation allows both factor Xa to bind to released platelet factor V(a) and makes available a surface membrane component, probably anionic phospholipids, with which the bound factor Xa interacts.

Download Full-text

Human Platelets Contain Forms of Factor V in Disulfide-Linkage with Multimerin.

Blood ◽

10.1182/blood.v104.11.1933.1933 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1933-1933

Author(s):

Catherine P.M. Hayward ◽

Nola Fuller ◽

Shilun Zheng ◽

Frederic Adam ◽

Samira Jeimy ◽

...

Keyword(s):

Factor V ◽

Single Chain ◽

Platelet Factor ◽

Platelet Surface ◽

Disulfide Linkage ◽

Thrombin Cleavage ◽

Factor Va ◽

Plasma Factor ◽

Large Factor ◽

V Antigen

Abstract Factor V is an essential cofactor for blood coagulation that circulates in platelets and plasma. Unlike plasma factor V, platelet factor V is stored complexed with the polymeric α-granule protein multimerin. In analyses of human platelet factor V on nonreduced denaturing multimer gels, we identified that approximately 25% was variable in size and migrated larger than single chain factor V, the largest form in plasma. Upon reduction, the unusually large, variably-sized forms of platelet factor V liberated components that comigrated with other forms of platelet factor V, indicating that they contained factor V in interchain disulfide-linkages. With thrombin cleavage, factor Va heavy and light chain domains, but not B-domains, were liberated from the components linked by interchain disulfide bonds, indicating that the single cysteine in the B-domain at position 1085 was the site of disulfide linkage. Because unusually large factor V had a variable size and included forms larger than factor V dimers, the data suggested disulfide-linkage with another platelet protein, possibly multimerin. Immunoprecipitation experiments confirmed that all unusually large factor V in platelets was associated with multimerin and it remained associated in 0.5 M salt. Multimerin immunodepletion of the normal pooled platelet lysate removed 100 ± 0% of multimerin and 47.0 ± 2.4% of total factor V antigen, whereas sham immunodepletion removed 12.0 ± 3.0 % of multimerin and 4.0 ± 4.0% of factor V antigen (means ± 1 S.D. for 3 experiments). Analyses of serial factor V immunopurified samples indicated that platelets contained a subpopulation of multimerin polymers that resisted dissociation from factor V by denaturing detergent and comigrated with unusually large platelet factor V, before and after thrombin cleavage. The suggestion that only a subpopulation of multimerin was covalently linked to factor V was consistent with the estimated 17 fold molar excess of multimerin subunits to factor V molecules in platelets. The disulfide-linked complexes of multimerin and factor V in platelets, that are cleaved by thrombin to liberate factor Va, could be important for modulating the function of platelet factor V and its delivery onto activated platelets. Multimerin could function to hold about half of the platelet pool of factor V in covalent and noncovalent linkages, until granule release occurs and thrombin cleavages liberate factor Va for prothrombinase assembly on the platelet surface, akin to the way supporting scaffolds hold pieces of plastic models in a unit until their removal for model assembly is desired.

Download Full-text

von Willebrand factor is present on the surface of platelets stimulated in plasma by ADP

Blood ◽

10.1182/blood.v70.5.1362.bloodjournal7051362 ◽

1987 ◽

Vol 70 (5) ◽

pp. 1362-1366

Author(s):

B Adelman ◽

P Carlson ◽

P Powers

Keyword(s):

Binding Site ◽

Von Willebrand Factor ◽

Platelet Rich Plasma ◽

Calcium Ionophore ◽

Calcium Ionophore A23187 ◽

Ionophore A23187 ◽

Surface Binding ◽

Platelet Surface ◽

Von Willebrand ◽

Willebrand Factor

von Willebrand factor (vWf) can bind to glycoprotein (GP) IIb/IIIa on activated platelets. The significance of this interaction is unclear, however, because it has not been possible to detect vWf binding to GPIIb/IIIa on platelets stimulated in plasma. We have developed an indirect, flow cytometry assay that uses fluorescein-labeled antibodies to detect vWf and fibrinogen on platelets. Using this assay, we found vWf on the surface of platelets stimulated in plasma by ADP. The number of platelets that bound vWf increased in proportion to ADP concentration and incubation time. Washed platelets in a protein-free buffer activated by 1 mumol/L calcium ionophore A23187 or 10 mumol/L ADP also bound vWf, suggesting that we were detecting surface binding of alpha-granule-derived vWf. Monoclonal antibodies against the vWf binding site on GPIb (6D1) and the vWf and fibrinogen binding sites on GPIIb/IIIa (LJP5 and LJ-CP8, respectively) were used to characterize the mechanism of vWf binding to stimulated platelets. Ristocetin- induced binding of vWf was inhibited by 6D1, and ADP-induced binding of fibrinogen was inhibited by LJ-CP8. None of these antibodies inhibited ADP-induced vWf binding. Aspirin and prostaglandin E1 also inhibited ADP-induced binding of vWf in platelet-rich plasma. During platelet activation in plasma, vWf derived from alpha-granules becomes bound to the platelet surface possibly being transferred already associated with a binding site.

Download Full-text

The effect of platelets upon factor Xa-catalyzed activation of factor VII in vitro

Blood ◽

10.1182/blood.v72.2.396.bloodjournal722396 ◽

1988 ◽

Vol 72 (2) ◽

pp. 396-401

Author(s):

LV Rao ◽

SI Rapaport

Keyword(s):

Surface Membrane ◽

Calcium Ionophore ◽

Factor Xa ◽

Factor Vii ◽

Ionophore A23187 ◽

Exogenous Factor ◽

Platelet Factor ◽

Anionic Phospholipids ◽

Factor Va ◽

Activated Platelets

The authors have investigated the ability of platelets to enhance factor Xa-catalyzed activation of factor VII. Unstimulated platelets were without effect, whereas freeze/thawed platelets substantially enhanced activation. Antifactor V antibodies did not diminish the enhancement. Platelets activated by thrombin, collagen, or calcium ionophore A23187 also enhanced factor Xa-catalyzed activation of factor VII. In contrast to their lack of effect upon freeze/thawed platelets, antifactor V antibodies abolished augmented factor VII activation induced by activated platelets. Adding exogenous factor Va to unstimulated platelets failed to enhance factor Xa-catalyzed activation of factor VII, nor did adding exogenous factor Va to activated platelets augment activation beyond that observed with activated platelets alone. These observations can be interpreted as follows: (1) factor Va does not function as a cofactor for factor Xa-catalyzed activation of factor VII; (2) anionic phospholipids are a known cofactor for factor Xa-catalyzed activation of factor VII, and freeze/thawed platelets probably enhance activation by making anionic phospholipids on disrupted platelet membranes available to function as a cofactor; (3) the presumed binding of factor Xa to exogenous factor Va on unstimulated platelets is insufficient in itself to augment factor Xa-catalyzed activation of factor VII; (4) activated platelets augment factor Xa-catalyzed factor VII activation because activation allows both factor Xa to bind to released platelet factor V(a) and makes available a surface membrane component, probably anionic phospholipids, with which the bound factor Xa interacts.

Download Full-text