Thrombin

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Johan W M Heemskerk - One of the best experts on this subject based on the ideXlab platform.

  • rate limiting roles of the tenase complex of factors viii and ix in platelet procoagulant activity and formation of platelet fibrin thrombi under flow
    Haematologica, 2015
    Co-Authors: Frauke Swieringa, Marijke J E Kuijpers, Moniek M E Lamers, Paola E J Van Der Meijden, Johan W M Heemskerk
    Abstract:

    The importance of factor Xa generation in thrombus formation has not been studied extensively so far. Here, we used mice deficient in either factor VIII or factor IX to determine the role of platelet-stimulated tenase activity in the formation of platelet-fibrin thrombi on collagen. With tissue factor present, deficiency in factor VIII or IX markedly suppressed thrombus growth, fibrin formation and platelet procoagulant activity (phosphatidylserine exposure). In either case, residual fibrin formation was eliminated in the absence of tissue factor. Effects of factor deficiencies were antagonized by supplementation of the missing coagulation factor. In wild-type thrombi generated under flow, phosphatidylserine-exposing platelets bound (activated) factor IX and factor X, whereas factor VIII preferentially co-localized at sites of von Willebrand factor binding. Furthermore, proteolytic activity of the generated activated factor X and Thrombin was confined to the sites of phosphatidylserine exposure. With blood from a hemophilia A or B patient, the formation of platelet-fibrin thrombi was greatly delayed and reduced, even in the presence of high concentrations of tissue factor. A direct activated factor X inhibitor, rivaroxaban, added to human blood, suppressed both Thrombin and fibrin formation. Together, these data point to a potent enforcement loop in thrombus formation due to factor X activation, subsequent Thrombin and fibrin generation, causing activated factor X-mediated stimulation of platelet phosphatidylserine exposure. This implies that the factor VIII/factor IX-dependent stimulation of platelet procoagulant activity is a limiting factor for fibrin formation under flow conditions, even at high tissue factor concentrations.

  • roles of platelet stim1 and orai1 in glycoprotein vi and Thrombin dependent procoagulant activity and thrombus formation
    Journal of Biological Chemistry, 2010
    Co-Authors: Karen Gilio, Marijke J E Kuijpers, Paola E J Van Der Meijden, Roger Van Kruchten, Attila Braun, Alejandro Bernaerro, Marion A H Feijge, David Stegner, David Vargaszabo, Johan W M Heemskerk
    Abstract:

    In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca2+ entry (SOCE) with Orai1 as principal Ca2+ entry channel. Both proteins contribute to collagen-dependent arterial thrombosis in mice in vivo. It is unclear whether STIM2 is involved. A key platelet response relying on Ca2+ entry is the surface exposure of phosphatidylserine (PS), which accomplishes platelet procoagulant activity. We studied this response in mouse platelets deficient in STIM1, STIM2, or Orai1. Upon high shear flow of blood over collagen, Stim1−/− and Orai1−/− platelets had greatly impaired glycoprotein (GP) VI-dependent Ca2+ signals, and they were deficient in PS exposure and thrombus formation. In contrast, Stim2−/− platelets reacted normally. Upon blood flow in the presence of Thrombin generation and coagulation, Ca2+ signals of Stim1−/− and Orai1−/− platelets were partly reduced, whereas the PS exposure and formation of fibrin-rich thrombi were normalized. Washed Stim1−/− and Orai1−/− platelets were deficient in GPVI-induced PS exposure and proThrombinase activity, but not when Thrombin was present as co-agonist. Markedly, SKF96365, a blocker of (receptor-operated) Ca2+ entry, inhibited Ca2+ and procoagulant responses even in Stim1−/− and Orai1−/− platelets. These data show for the first time that: (i) STIM1 and Orai1 jointly contribute to GPVI-induced SOCE, procoagulant activity, and thrombus formation; (ii) a compensating Ca2+ entry pathway is effective in the additional presence of Thrombin; (iii) platelets contain two mechanisms of Ca2+ entry and PS exposure, only one relying on STIM1-Orai1 interaction.

  • key role of platelet procoagulant activity in tissue factor and collagen dependent thrombus formation in arterioles and venules in vivo differential sensitivity to Thrombin inhibition
    Microcirculation, 2008
    Co-Authors: Marijke J E Kuijpers, Imke C A Munnix, Judith M E M Cosemans, Bart J M Van Vlijmen, Chris P M Reutelingsperger, Mirjam Oude G A Egbrink, Johan W M Heemskerk
    Abstract:

    Objective: Blood coagulation and platelet activation are mutually dependent processes, but contribute differently to venous and arterial thrombosis. We investigated the interplay of these processes in vivo in a mouse model of arteriolar and venular thrombus formation.Methods: Thrombus formation was studied by intravital (fluorescence) microscopy after topical application of FeCl3 on mouse mesenteric microvessels.Results: Both in arterioles and venules, the thrombus-forming process relied on tissue factor-factor VII(a) interaction, collagen exposure, and glycoprotein VI–mediated platelet activation. Arterial thrombus formation was impaired by mild Thrombin inhibition or platelet inhibition, while venous thrombosis was only suppressed by strong Thrombin inhibition or by mild Thrombin inhibition together with platelet inhibition. Phosphatidylserine-exposing platelets were present in thrombi of both vessel types, as detected with fluorescently labeled annexin A5. Injection of annexin A5 to shield exposed phos...

  • dual role of platelet protein kinase c in thrombus formation stimulation of pro aggregatory and suppression of procoagulant activity in platelets
    Journal of Biological Chemistry, 2007
    Co-Authors: Amrei Strehl, Marijke J E Kuijpers, Paola E J Van Der Meijden, Imke C A Munnix, Judith M E M Cosemans, Marion A H Feijge, Bernhard Nieswandt, Johan W M Heemskerk
    Abstract:

    Abstract Protein kinase C (PKC) isoforms regulate many platelet responses in a still incompletely understood manner. Here we investigated the roles of PKC in the platelet reactions implicated in thrombus formation as follows: secretion aggregate formation and coagulation-stimulating activity, using inhibitors with proven activity in plasma. In human and mouse platelets, PKC regulated aggregation by mediating secretion and contributing to αIIbβ3 activation. Strikingly, PKC suppressed Ca2+ signal generation and Ca2+-dependent exposure of procoagulant phosphatidylserine. Furthermore, under coagulant conditions, PKC suppressed the Thrombin-generating capacity of platelets. In flowing human and mouse blood, PKC contributed to platelet adhesion and controlled secretion-dependent thrombus formation, whereas it down-regulated Ca2+ signaling and procoagulant activity. In murine platelets lacking Gqα, where secretion reactions were reduced in comparison with wild type mice, PKC still positively regulated platelet aggregation and down-regulated procoagulant activity. We conclude that platelet PKC isoforms have a dual controlling role in thrombus formation as follows: (i) by mediating secretion and integrin activation required for platelet aggregation under flow, and (ii) by suppressing Ca2+-dependent phosphatidylserine exposure, and consequently Thrombin generation and coagulation. This platelet signaling protein is the first one identified to balance the pro-aggregatory and procoagulant functions of thrombi.

Marijke J E Kuijpers - One of the best experts on this subject based on the ideXlab platform.

  • rate limiting roles of the tenase complex of factors viii and ix in platelet procoagulant activity and formation of platelet fibrin thrombi under flow
    Haematologica, 2015
    Co-Authors: Frauke Swieringa, Marijke J E Kuijpers, Moniek M E Lamers, Paola E J Van Der Meijden, Johan W M Heemskerk
    Abstract:

    The importance of factor Xa generation in thrombus formation has not been studied extensively so far. Here, we used mice deficient in either factor VIII or factor IX to determine the role of platelet-stimulated tenase activity in the formation of platelet-fibrin thrombi on collagen. With tissue factor present, deficiency in factor VIII or IX markedly suppressed thrombus growth, fibrin formation and platelet procoagulant activity (phosphatidylserine exposure). In either case, residual fibrin formation was eliminated in the absence of tissue factor. Effects of factor deficiencies were antagonized by supplementation of the missing coagulation factor. In wild-type thrombi generated under flow, phosphatidylserine-exposing platelets bound (activated) factor IX and factor X, whereas factor VIII preferentially co-localized at sites of von Willebrand factor binding. Furthermore, proteolytic activity of the generated activated factor X and Thrombin was confined to the sites of phosphatidylserine exposure. With blood from a hemophilia A or B patient, the formation of platelet-fibrin thrombi was greatly delayed and reduced, even in the presence of high concentrations of tissue factor. A direct activated factor X inhibitor, rivaroxaban, added to human blood, suppressed both Thrombin and fibrin formation. Together, these data point to a potent enforcement loop in thrombus formation due to factor X activation, subsequent Thrombin and fibrin generation, causing activated factor X-mediated stimulation of platelet phosphatidylserine exposure. This implies that the factor VIII/factor IX-dependent stimulation of platelet procoagulant activity is a limiting factor for fibrin formation under flow conditions, even at high tissue factor concentrations.

  • roles of platelet stim1 and orai1 in glycoprotein vi and Thrombin dependent procoagulant activity and thrombus formation
    Journal of Biological Chemistry, 2010
    Co-Authors: Karen Gilio, Marijke J E Kuijpers, Paola E J Van Der Meijden, Roger Van Kruchten, Attila Braun, Alejandro Bernaerro, Marion A H Feijge, David Stegner, David Vargaszabo, Johan W M Heemskerk
    Abstract:

    In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca2+ entry (SOCE) with Orai1 as principal Ca2+ entry channel. Both proteins contribute to collagen-dependent arterial thrombosis in mice in vivo. It is unclear whether STIM2 is involved. A key platelet response relying on Ca2+ entry is the surface exposure of phosphatidylserine (PS), which accomplishes platelet procoagulant activity. We studied this response in mouse platelets deficient in STIM1, STIM2, or Orai1. Upon high shear flow of blood over collagen, Stim1−/− and Orai1−/− platelets had greatly impaired glycoprotein (GP) VI-dependent Ca2+ signals, and they were deficient in PS exposure and thrombus formation. In contrast, Stim2−/− platelets reacted normally. Upon blood flow in the presence of Thrombin generation and coagulation, Ca2+ signals of Stim1−/− and Orai1−/− platelets were partly reduced, whereas the PS exposure and formation of fibrin-rich thrombi were normalized. Washed Stim1−/− and Orai1−/− platelets were deficient in GPVI-induced PS exposure and proThrombinase activity, but not when Thrombin was present as co-agonist. Markedly, SKF96365, a blocker of (receptor-operated) Ca2+ entry, inhibited Ca2+ and procoagulant responses even in Stim1−/− and Orai1−/− platelets. These data show for the first time that: (i) STIM1 and Orai1 jointly contribute to GPVI-induced SOCE, procoagulant activity, and thrombus formation; (ii) a compensating Ca2+ entry pathway is effective in the additional presence of Thrombin; (iii) platelets contain two mechanisms of Ca2+ entry and PS exposure, only one relying on STIM1-Orai1 interaction.

  • key role of platelet procoagulant activity in tissue factor and collagen dependent thrombus formation in arterioles and venules in vivo differential sensitivity to Thrombin inhibition
    Microcirculation, 2008
    Co-Authors: Marijke J E Kuijpers, Imke C A Munnix, Judith M E M Cosemans, Bart J M Van Vlijmen, Chris P M Reutelingsperger, Mirjam Oude G A Egbrink, Johan W M Heemskerk
    Abstract:

    Objective: Blood coagulation and platelet activation are mutually dependent processes, but contribute differently to venous and arterial thrombosis. We investigated the interplay of these processes in vivo in a mouse model of arteriolar and venular thrombus formation.Methods: Thrombus formation was studied by intravital (fluorescence) microscopy after topical application of FeCl3 on mouse mesenteric microvessels.Results: Both in arterioles and venules, the thrombus-forming process relied on tissue factor-factor VII(a) interaction, collagen exposure, and glycoprotein VI–mediated platelet activation. Arterial thrombus formation was impaired by mild Thrombin inhibition or platelet inhibition, while venous thrombosis was only suppressed by strong Thrombin inhibition or by mild Thrombin inhibition together with platelet inhibition. Phosphatidylserine-exposing platelets were present in thrombi of both vessel types, as detected with fluorescently labeled annexin A5. Injection of annexin A5 to shield exposed phos...

  • dual role of platelet protein kinase c in thrombus formation stimulation of pro aggregatory and suppression of procoagulant activity in platelets
    Journal of Biological Chemistry, 2007
    Co-Authors: Amrei Strehl, Marijke J E Kuijpers, Paola E J Van Der Meijden, Imke C A Munnix, Judith M E M Cosemans, Marion A H Feijge, Bernhard Nieswandt, Johan W M Heemskerk
    Abstract:

    Abstract Protein kinase C (PKC) isoforms regulate many platelet responses in a still incompletely understood manner. Here we investigated the roles of PKC in the platelet reactions implicated in thrombus formation as follows: secretion aggregate formation and coagulation-stimulating activity, using inhibitors with proven activity in plasma. In human and mouse platelets, PKC regulated aggregation by mediating secretion and contributing to αIIbβ3 activation. Strikingly, PKC suppressed Ca2+ signal generation and Ca2+-dependent exposure of procoagulant phosphatidylserine. Furthermore, under coagulant conditions, PKC suppressed the Thrombin-generating capacity of platelets. In flowing human and mouse blood, PKC contributed to platelet adhesion and controlled secretion-dependent thrombus formation, whereas it down-regulated Ca2+ signaling and procoagulant activity. In murine platelets lacking Gqα, where secretion reactions were reduced in comparison with wild type mice, PKC still positively regulated platelet aggregation and down-regulated procoagulant activity. We conclude that platelet PKC isoforms have a dual controlling role in thrombus formation as follows: (i) by mediating secretion and integrin activation required for platelet aggregation under flow, and (ii) by suppressing Ca2+-dependent phosphatidylserine exposure, and consequently Thrombin generation and coagulation. This platelet signaling protein is the first one identified to balance the pro-aggregatory and procoagulant functions of thrombi.

Alisa S Wolberg - One of the best experts on this subject based on the ideXlab platform.

  • fibrinogen and red blood cells in venous thrombosis
    Thrombosis Research, 2014
    Co-Authors: Maria M Aleman, Bethany L Walton, James R Byrnes, Alisa S Wolberg
    Abstract:

    Deep vein thrombosis and pulmonary embolism, collectively termed venous thromboembolism (VTE), affect over 1 million Americans each year. VTE is triggered by inflammation and blood stasis leading to the formation of thrombi rich in fibrin and red blood cells (RBCs). However, little is known about mechanisms regulating fibrin and RBC incorporation into venous thrombi, or how these components mediate thrombus size or resolution. Both elevated circulating fibrinogen (hyperfibrinogenemia) and abnormal fibrin(ogen) structure and function, including increased fibrin network density and resistance to fibrinolysis, have been observed in plasmas from patients with VTE. Abnormalities in RBC number and/or function have also been associated with VTE risk. RBC contributions to VTE are thought to stem from their effects on blood viscosity and margination of platelets to the vessel wall. More recent studies suggest RBCs also express phosphatidylserine, support Thrombin generation, and decrease fibrinolysis. RBC interactions with fibrin(ogen) and cells, including platelets and endothelial cells, may also promote thrombus formation. The contributions of fibrin(ogen) and RBCs to the pathophysiology of VTE warrants further investigation.

  • procoagulant activity induced by vascular injury determines contribution of elevated factor viii to thrombosis and thrombus stability in mice
    Blood, 2011
    Co-Authors: Kellie R Machlus, Alisa S Wolberg
    Abstract:

    Studies have correlated elevated plasma factor VIII (FVIII) with thrombosis; however, it is unclear whether elevated FVIII is a proinflammatory biomarker, causative agent, or both. We raised FVIII levels in mice and measured the time to vessel occlusion (TTO) after ferric chloride–induced injury. Compared with control (saline-infused) mice, elevated FVIII had no effect after longer (3-minute) carotid artery injury, but it shortened the TTO after shorter (2-minute) injury (P < .008). After injury, circulating Thrombin-antiThrombin (TAT) complexes were lower after short versus long injury (P < .04), suggesting short treatment produced less coagulation activation. TAT levels in FVIII-infused mice were higher than in controls after short, but not longer, injury. Accordingly, elevated FVIII had no effect on in vitro Thrombin generation or platelet aggregation triggered by high tissue factor, but it increased Thrombin generation rate and peak (2.4- and 1.5-fold, respectively), and it accelerated platelet aggregation (up to 1.6-fold) when initiated by low tissue factor. Compared with control mice, elevated FVIII stabilized thrombi (fewer emboli) after short injury, but it had no effect after longer injury. TTO and emboli correlated with TATs. These results demonstrate dependence of FVIII activity on extent of vascular injury. We propose elevated plasma FVIII is an etiologic, prothrombotic agent after moderate but not extensive vascular damage.

Paola E J Van Der Meijden - One of the best experts on this subject based on the ideXlab platform.

  • rate limiting roles of the tenase complex of factors viii and ix in platelet procoagulant activity and formation of platelet fibrin thrombi under flow
    Haematologica, 2015
    Co-Authors: Frauke Swieringa, Marijke J E Kuijpers, Moniek M E Lamers, Paola E J Van Der Meijden, Johan W M Heemskerk
    Abstract:

    The importance of factor Xa generation in thrombus formation has not been studied extensively so far. Here, we used mice deficient in either factor VIII or factor IX to determine the role of platelet-stimulated tenase activity in the formation of platelet-fibrin thrombi on collagen. With tissue factor present, deficiency in factor VIII or IX markedly suppressed thrombus growth, fibrin formation and platelet procoagulant activity (phosphatidylserine exposure). In either case, residual fibrin formation was eliminated in the absence of tissue factor. Effects of factor deficiencies were antagonized by supplementation of the missing coagulation factor. In wild-type thrombi generated under flow, phosphatidylserine-exposing platelets bound (activated) factor IX and factor X, whereas factor VIII preferentially co-localized at sites of von Willebrand factor binding. Furthermore, proteolytic activity of the generated activated factor X and Thrombin was confined to the sites of phosphatidylserine exposure. With blood from a hemophilia A or B patient, the formation of platelet-fibrin thrombi was greatly delayed and reduced, even in the presence of high concentrations of tissue factor. A direct activated factor X inhibitor, rivaroxaban, added to human blood, suppressed both Thrombin and fibrin formation. Together, these data point to a potent enforcement loop in thrombus formation due to factor X activation, subsequent Thrombin and fibrin generation, causing activated factor X-mediated stimulation of platelet phosphatidylserine exposure. This implies that the factor VIII/factor IX-dependent stimulation of platelet procoagulant activity is a limiting factor for fibrin formation under flow conditions, even at high tissue factor concentrations.

  • roles of platelet stim1 and orai1 in glycoprotein vi and Thrombin dependent procoagulant activity and thrombus formation
    Journal of Biological Chemistry, 2010
    Co-Authors: Karen Gilio, Marijke J E Kuijpers, Paola E J Van Der Meijden, Roger Van Kruchten, Attila Braun, Alejandro Bernaerro, Marion A H Feijge, David Stegner, David Vargaszabo, Johan W M Heemskerk
    Abstract:

    In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca2+ entry (SOCE) with Orai1 as principal Ca2+ entry channel. Both proteins contribute to collagen-dependent arterial thrombosis in mice in vivo. It is unclear whether STIM2 is involved. A key platelet response relying on Ca2+ entry is the surface exposure of phosphatidylserine (PS), which accomplishes platelet procoagulant activity. We studied this response in mouse platelets deficient in STIM1, STIM2, or Orai1. Upon high shear flow of blood over collagen, Stim1−/− and Orai1−/− platelets had greatly impaired glycoprotein (GP) VI-dependent Ca2+ signals, and they were deficient in PS exposure and thrombus formation. In contrast, Stim2−/− platelets reacted normally. Upon blood flow in the presence of Thrombin generation and coagulation, Ca2+ signals of Stim1−/− and Orai1−/− platelets were partly reduced, whereas the PS exposure and formation of fibrin-rich thrombi were normalized. Washed Stim1−/− and Orai1−/− platelets were deficient in GPVI-induced PS exposure and proThrombinase activity, but not when Thrombin was present as co-agonist. Markedly, SKF96365, a blocker of (receptor-operated) Ca2+ entry, inhibited Ca2+ and procoagulant responses even in Stim1−/− and Orai1−/− platelets. These data show for the first time that: (i) STIM1 and Orai1 jointly contribute to GPVI-induced SOCE, procoagulant activity, and thrombus formation; (ii) a compensating Ca2+ entry pathway is effective in the additional presence of Thrombin; (iii) platelets contain two mechanisms of Ca2+ entry and PS exposure, only one relying on STIM1-Orai1 interaction.

  • dual role of platelet protein kinase c in thrombus formation stimulation of pro aggregatory and suppression of procoagulant activity in platelets
    Journal of Biological Chemistry, 2007
    Co-Authors: Amrei Strehl, Marijke J E Kuijpers, Paola E J Van Der Meijden, Imke C A Munnix, Judith M E M Cosemans, Marion A H Feijge, Bernhard Nieswandt, Johan W M Heemskerk
    Abstract:

    Abstract Protein kinase C (PKC) isoforms regulate many platelet responses in a still incompletely understood manner. Here we investigated the roles of PKC in the platelet reactions implicated in thrombus formation as follows: secretion aggregate formation and coagulation-stimulating activity, using inhibitors with proven activity in plasma. In human and mouse platelets, PKC regulated aggregation by mediating secretion and contributing to αIIbβ3 activation. Strikingly, PKC suppressed Ca2+ signal generation and Ca2+-dependent exposure of procoagulant phosphatidylserine. Furthermore, under coagulant conditions, PKC suppressed the Thrombin-generating capacity of platelets. In flowing human and mouse blood, PKC contributed to platelet adhesion and controlled secretion-dependent thrombus formation, whereas it down-regulated Ca2+ signaling and procoagulant activity. In murine platelets lacking Gqα, where secretion reactions were reduced in comparison with wild type mice, PKC still positively regulated platelet aggregation and down-regulated procoagulant activity. We conclude that platelet PKC isoforms have a dual controlling role in thrombus formation as follows: (i) by mediating secretion and integrin activation required for platelet aggregation under flow, and (ii) by suppressing Ca2+-dependent phosphatidylserine exposure, and consequently Thrombin generation and coagulation. This platelet signaling protein is the first one identified to balance the pro-aggregatory and procoagulant functions of thrombi.

Ningsheng Shao - One of the best experts on this subject based on the ideXlab platform.

  • RNA aptamers specific for bovine Thrombin
    Journal of Molecular Recognition, 2003
    Co-Authors: Xuemei Liu, Guojun Cao, Hongmei Ding, Ming Fan, Dajin Zhang, Beifen Shen, Gang Liu, Guang Yang, Ningsheng Shao
    Abstract:

    Bovine Thrombin is widely used in clinical wound healing after surgery. There is 85% homology between bovine Thrombin and human Thrombin, so most antibodies against bovine Thrombin cross-react with human Thrombin. Rare antibodies against bovine Thrombin but not cross-reacting with human Thrombin have been reported. RNA ligands (aptamers) have been used to bind to target molecules with sometimes higher specificity than antibodies. Here we report the isolation of aptamers specific for bovine Thrombin by systematic evolution of ligands by exponential enrichment (SELEX) from an RNA pool containing a 25-nucleotide randomized region. After seven rounds of selection, two aptamers specific for bovine Thrombin were identified with a K-d of 164 and 240 nM, respectively. Significantly, these aptamers do not bind to human Thrombin. Secondary structure prediction revealed potential stem-loop structures for these RNAs. Both RNA aptamers inhibit only bovine Thrombin-catalyzed fibrin clot formation in vitro. Competition assay results suggested that the RNA aptamers might bind to the electropositive domain of bovine Thrombin, that is, heparin-binding site, instead of fibrinogen-recognition exosite. The resulting bovine-specific Thrombin inhibitor might be used in some clinical applications when bovine Thrombin activity needs to be contained or in research where human and bovine Thrombin need to be distinguished. Copyright (C) 2003 John Wiley Sons, Ltd.