Thrombin: A Pivotal Player in Hemostasis and Beyond

2021 ◽  
Author(s):  
Julie Brogaard Larsen ◽  
Anne-Mette Hvas
Keyword(s):  
Thrombin Generation ◽  
Ex Vivo ◽  
Coagulation Factor ◽  
Fluid Phase ◽  
Fibrin Clot ◽  
Indirect Measurement ◽  
Protease Activated Receptors ◽  
Platelet Surface ◽  
Thromboembolic Risk

AbstractThe serine protease thrombin, a naturally derived enzyme, plays a key role in hemostasis by converting fibrinogen to fibrin and activating coagulation factor XIII whereby the fibrin clot is stabilized. Furthermore, thrombin activates platelets through protease-activated receptors on the platelet surface. Conversely, thrombin also exerts anticoagulant effects, enhancing the protein C activity while complexed with thrombomodulin. During recent years, it has become evident that thrombin has significant effects beyond hemostasis, as it contributes also to modulation of the endothelium, promotes inflammation and angiogenesis, and plays a role in tumor progression. Yet, due to the very short half-life and almost immediate inhibition in fluid phase by antithrombin, thrombin itself remains elusive, and only indirect measurement of thrombin generation is possible. This review provides a description of structure and mechanisms of action of thrombin both in physiological and pathological processes. Furthermore, it summarizes laboratory tests that measure in vivo or ex vivo thrombin generation, and presents knowledge on the value of these biomarkers in bleeding disorders, cardiopulmonary bypass surgery, and thromboembolic risk assessment in different patient populations. Finally, this review outlines further perspectives on using thrombin generation biomarkers for research purposes and in clinical practice.

Factor XIIa regulates the structure of the fibrin clot independently of thrombin generation through direct interaction with fibrin

Blood ◽  
2011 ◽  
Vol 118 (14) ◽  
pp. 3942-3951 ◽  
Author(s):  
Joke Konings ◽  
José W. P. Govers-Riemslag ◽  
Helen Philippou ◽  
Nicola J. Mutch ◽  
Julian I. Borissoff ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Thrombus Formation ◽  
Coagulation Factor ◽  
Direct Interaction ◽  
Fibrin Clot ◽  
Fiber Density ◽  
Contact Pathway ◽  
Clot Structure ◽  
Fibrin Structure

Abstract Recent data indicate an important contribution of coagulation factor (F)XII to in vivo thrombus formation. Because fibrin structure plays a key role in clot stability and thrombosis, we hypothesized that FXII(a) interacts with fibrin(ogen) and thereby regulates clot structure and function. In plasma and purified system, we observed a dose-dependent increase in fibrin fiber density and decrease in turbidity, reflecting a denser structure, and a nonlinear increase in clot stiffness with FXIIa. In plasma, this increase was partly independent of thrombin generation, as shown in clots made in prothrombin-deficient plasma initiated with snake venom enzyme and in clots made from plasma deficient in FXII and prothrombin. Purified FXII and α-FXIIa, but not β-FXIIa, bound to purified fibrinogen and fibrin with nanomolar affinity. Immunostaining of human carotid artery thrombi showed that FXII colocalized with areas of dense fibrin deposition, providing evidence for the in vivo modulation of fibrin structure by FXIIa. These data demonstrate that FXIIa modulates fibrin clot structure independently of thrombin generation through direct binding of the N-terminus of FXIIa to fibrin(ogen). Modification of fibrin structure by FXIIa represents a novel physiologic role for the contact pathway that may contribute to the pathophysiology of thrombosis.

Ancylostoma caninum Anticoagulant Peptide Blocks Metastasis In Vivo and Inhibits Factor Xa Binding to Melanoma Cells In Vitro

1998 ◽  
Vol 79 (05) ◽  
pp. 1041-1047 ◽  
Author(s):  
Kathleen M. Donnelly ◽  
Michael E. Bromberg ◽  
Aaron Milstone ◽  
Jennifer Madison McNiff ◽  
Gordon Terwilliger ◽  
...  
Keyword(s):  
Active Site ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Melanoma Cells ◽  
Factor V ◽  
Ancylostoma Caninum ◽  
Metastatic Activity

SummaryWe evaluated the in vivo anti-metastatic activity of recombinant Ancylostoma caninum Anticoagulant Peptide (rAcAP), a potent (Ki = 265 pM) and specific active site inhibitor of human coagulation factor Xa originally isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with rAcAP (0.01-0.2 mg/mouse) prior to tail vein injection of LOX human melanoma cells resulted in a dose dependent reduction in pulmonary metastases. In order to elucidate potential mechanisms of rAcAP’s anti-metastatic activity, experiments were carried out to identify specific interactions between factor Xa and LOX. Binding of biotinylated factor Xa to LOX monolayers was both specific and saturable (Kd = 15 nM). Competition experiments using antibodies to previously identified factor Xa binding proteins, including factor V/Va, effector cell protease receptor-1, and tissue factor pathway inhibitor failed to implicate any of these molecules as significant binding sites for Factor Xa. Functional prothrombinase activity was also supported by LOX, with a half maximal rate of thrombin generation detected at a factor Xa concentration of 2.4 nM. Additional competition experiments using an excess of either rAcAP or active site blocked factor Xa (EGR-Xa) revealed that most of the total factor Xa binding to LOX is mediated via interaction with the enzyme’s active site, predicting that the vast majority of cell-associated factor Xa does not participate directly in thrombin generation. In addition to establishing two distinct mechanisms of factor Xa binding to melanoma, these data raise the possibility that rAcAP’s antimetastatic effect in vivo might involve novel non-coagulant pathways, perhaps via inhibition of active-site mediated interactions between factor Xa and tumor cells.

RPR120844, a Novel, Specific Inhibitor of Coagulation Factor Xa Inhibits Venous Thrombosis in the Rabbit

1999 ◽  
Vol 81 (01) ◽  
pp. 157-160 ◽  
Author(s):  
Ross Bentley ◽  
Suzanne Morgan ◽  
Karen Brown ◽  
Valeria Chu ◽  
Richard Ewing ◽  
...  
Keyword(s):  
Jugular Vein ◽  
Drug Effect ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Specific Inhibitor ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Antithrombotic Activity ◽  
Fxa Inhibitor

SummaryThe in vivo antithrombotic activity of RPR120844, a novel synthetic coagulation factor Xa (fXa) inhibitor (Ki = 7 nM), was assessed by its ability to inhibit thrombus formation in a damaged segment of the rabbit jugular vein. Intravenous dose-response studies were performed and thrombus mass (TM), activated partial thromboplastin time (APTT), prothrombin time (PT), inhibition of ex vivo fXa activity and plasma drug levels (PDL) were determined. TM, measured at the end of a 50 min infusion, was significantly reduced (p <0.05 vs saline-treated animals) by RPR120844 at 30 and 100 μg/kg/min. At doses of 10, 30 and 100 μg/kg/min, APTT was prolonged by 2.1, 4.2 and 6.1-fold, and PT was prolonged by 1.4, 2.2 and 3.5-fold, respectively. PDL were determined by measuring anti-fXa activity using an amidolytic assay. Peak PDL were 0.8 ± 0.3, 1.5 ± 0.9 and 2.4 ± 0.6 μM, respectively. The drug effect was reversible with APTT, PT and PDL returning toward pretreatment values 30 min after termination of treatment. The results suggest that RPR120844, or similar compounds, may provide an efficacious, yet easily reversible, means of inhibiting thrombus formation.

scholarly journals Protease-Activated Receptors 1 and 3 are Differentially Expressed on Human Monocyte Subsets and are Upregulated by Lipopolysaccharide Ex Vivo and In Vivo

2019 ◽  
Vol 119 (09) ◽  
pp. 1394-1402 ◽  
Author(s):  
Barbara Thaler ◽  
Philipp J. Hohensinner ◽  
Johanna Baumgartner ◽  
Patrick Haider ◽  
Konstantin A. Krychtiuk ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Critical Role ◽  
Human Monocyte ◽  
In Vivo Model ◽  
Monocyte Subsets ◽  
Plasminogen Activator Inhibitor 1 ◽  
Protease Activated Receptors ◽  
Messenger Ribonucleic Acid ◽  
Pai 1

AbstractMonocytes are activated in inflammatory conditions via a variety of cytokine receptors as well as in a procoagulatory setting through thrombin, acting upon protease-activated receptors (PARs). This study investigated the expression pattern of PAR1 and PAR3 on human monocyte subsets. Furthermore, a possible regulation of the expression of PAR1 and PAR3 in these cells by inflammatory activation were studied. CD16+ monocytes showed significantly higher levels of PAR1 and PAR3 as compared with CD16− monocytes. Ex vivo treatment of whole blood with lipopolysaccharide (LPS) increased PAR1 and PAR3 messenger ribonucleic acid (mRNA) in human monocytes. In addition, increase of PAR1 was seen in all three subsets upon LPS treatment, whereas PAR3 increased significantly only in CD16− monocytes and nonclassical CD16+ monocytes. Protein levels of PAR1 and PAR3 significantly increased on monocytes in vivo in human endotoxemia 1 hour after LPS infusion. PAR1 increased significantly in CD16− monocytes and nonclassical CD16+ monocytes. In this in vivo model, PAR3 was also significantly elevated in CD16− monocytes and increased slightly albeit not significantly in CD16+ monocytes. Endotoxemia increased plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF) expression in monocytes in humans. Pretreatment of healthy volunteers with the PAR1 antagonist vorapaxar blocked the increase in PAI-1 but not the increase in TF. We here provide new evidence for a critical role for monocytes as cellular mediators that contribute to the activation of coagulation in diseases characterized by an inflammatory state.

In Vivo and Ex Vivo Thrombin Generation in Noncomorbid Patients with Suspected Deep Venous Thrombosis

2019 ◽  
Vol 7 (2) ◽  
pp. 291-292
Author(s):  
Evi Kalodiki ◽  
Fredrik Wexels ◽  
Ola Dahl ◽  
Jeanine Walenga ◽  
Walter Jeske ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Deep Venous Thrombosis ◽  
Thrombin Generation ◽  
Ex Vivo

Interaction of Recombinant Factor VIIa with Rehydrated, Lyophilized Platelets.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3994-3994
Author(s):  
Thomas H. Fischer ◽  
Alisa S. Wolberg ◽  
Arthur P. Bode ◽  
Kevin J. Ramer ◽  
Timothy C. Nichols
Keyword(s):  
Surface Modification ◽  
Thrombin Generation ◽  
Factor Viia ◽  
Equilibrium Constants ◽  
Fluorescent Microscopy ◽  
Coagulation Factor ◽  
Platelet Membrane ◽  
Dependent Manner ◽  
Platelet Surface ◽  
Equilibrium Binding

Abstract The experiments presented here were undertaken to determine if factor VIIa (rFVIIa, the Novo Nordisk product NovoSeven™) will directly bind to rehydrated, lyophilized (RL) platelets (Stasix™ platelets, Entegrion, Inc. trade) for the formation of a catalytic surface with an enhanced ability to generate thrombin. The relationship of rFVIIa to the RL platelet surface was examined by measuring equilibrium and non-equilibrium binding of the coagulation factor to the cells, by studying the subcellular localization of the coagulation factor on RL platelets, and by following the effects of the surface modification on the kinetics of thrombin generation. The association of rFVIIa with RL platelets occurred with an on rate of 3.6x103 sec−1moles−1. Saturation occurred in minutes and was calcium dependent. Disassociation (in plasma or citrated saline) was slow, with over half of the coagulation factor remaining bound after two hours (with slow and fast rate constants of 5.0x10−5 and 4.1x10−4 sec−5 respectively). These results define a binding site with an apparent equilibrium constants of 110 nM. Equilibrium binding of rFVIIa to RL platelets was analyzed with flow cytometry and Western analysis. The rFVIIa was bound to RL platelets in a dose-dependent manner when incubated at concentrations of 0.3 to 10.0 uM rFVIIa and 3x104 to 106 RL platelets/ul in citrated saline. When high concentrations of rFVIIa were bound to RL platelets densities of over one million molecules of rFVIIa per RL platelet was obtained. Fluorescent microscopy analysis revealed that the rFVIIa was localized to the surface membrane and that some rFVIIa localized internally to the outer surface of the surface connected open canalicular system and/or sites of internal trafficking. Flow cytometric analysis with annexin V demonstrated that considerable quantities of phosphatidylserine were present on the external surface of the RL platelet membrane for potential facilitation of rFVIIa binding. The effect of RL platelet surface modification by rFVIIa on thrombin generation was investigated by following the hydrolysis of the thrombin-specific fluorogenic substate D-phe-pro-arg-ANSNHin plasma. rFVIIa and RL platelets accelerated thrombin generation in this system with rFVIIa being approximately twice as effective (per molecule of the recombinant protein) when added to the assay system pre-bound to RL platelets as compared to being initially free in the plasma. Similar results were obtained when free and RL platelet bound rFVIIa were tested in factor IX-deficient plasma. These experiments show that rFVIIa retains activity when super-saturated on the RL platelet membrane. The results of the studies presented here suggest that RL platelets can be used to concentrate rFVIIa at sites of vascular injury.

Feedback Activation of Factor XI by Thrombin Is Essential for Hemostasis In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2127-2127
Author(s):  
Henri M. H. Spronk ◽  
Sabine Wilhelm ◽  
Rene Van Oerle ◽  
Menno L. Knetsch ◽  
David Gailani ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Factor Viia ◽  
Fibrin Clot ◽  
Intrauterine Death ◽  
Factor Xii ◽  
Dependent Manner ◽  
Factor Xi ◽  
Feedback Activation

Abstract Abstract 2127 Poster Board II-102 Background: The revised model of coagulation proposes that factor XI (FXI) can be activated by thrombin, which is generated upon activation of the tissue factor (TF) pathway. This concept, however, has not been tested in vivo. A recent study questioned the existence of this feedback loop and suggested that factor XII (FXII) is the sole activator of FXI. Here, we analyze the feedback activation of FXI in plasma and in genetically altered mice. Methods and results: Fluorescence-based assays indicated that particle-bound thrombin caused thrombin generation in plasma both in the absence of TF and in the presence of active site inhibited factor VIIa. Thrombin failed to activate FXII and thrombin generation was almost completely abolished by an anti-FXIa antibody and in FXI-deficient plasma. Surface bound thrombin induced complex formation of FXI, with its major inhibitor C1 inhibitor, even in FXII-deficient plasma in a time and dose dependent manner. To determine if thrombin-driven FXI activation is important for hemostasis in vivo we used TF deficient mice (low TF), which have severely reduced thrombin formation. Low TF mice were crossed with mice deficient in one of the intrinsic pathway proteases FXII, FXI, or FIX. Double deficiency in TF and either FIX or FXI resulted in the intrauterine death of embryos due to hemorrhage. In contrast low TF/FXII-null mice were viable and the bleeding phenotype was unchanged from low TF animals. Conclusions: Surface-bound thrombin, a model for fibrin clot-protected thrombin, generates thrombin in a FXI dependent manner, independently from FXII. In addition to corroborating an amplifying role of FXI in thrombin generation, we provide the first evidence that at low TF levels FXI is essential in generating a sufficient ambient level of thrombin to permit embryonic development. Disclosures: No relevant conflicts of interest to declare.

Synergistic Effects of Bifunctional Antibodies Against beta3 Integrin on Dissolution of Platelet Thrombus,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3350-3350
Author(s):  
Wei Zhang ◽  
Suying Dang ◽  
Thomas Wisniewski
Keyword(s):  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Synergistic Effects ◽  
Fibrin Clot ◽  
Clot Lysis ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Platelet Thrombus ◽  
Platelet Aggregates

Abstract Abstract 3350 HIV-ITP patients have a unique Ab against platelet GPIIIa49-66 which induces oxidative platelet fragmentation in the absence of complement (Cell 106: 551, 2001; JCI 113: 973, 2004). Using a phage display single-chain antibody (scFv) library, we developed a novel human monoclonal scFv Ab against GPIIIa49-66 (named A11), which act similarly to the parental Ab (JBC 283: 3224, 2008). We then produced a bifunctional GPIIIa49-66 agent (named SLK), that targets newly deposited fibrin strands within and surrounding the platelet thrombus and has reduced effects on non-activated circulating platelets (Blood 116: 2336, 2010). In this study, we produced another bifunctional GPIIIa49-66 agent (named APAC), which homes to activated platelets. Like SLK, APAC destroys platelet aggregates ex vivo in an identical fashion with ∼85% destruction of platelet aggregates at 2 hrs. Platelet aggregate dissolution with a combination of SLK and APAC was ∼2 fold greater than either agent alone at 0.025 μM. Platelet-rich clot lysis experiments demonstrated the time required for 50% platelet-rich fibrin clot lysis (T50%) by APAC (95±6.1 min) was significantly longer than that by APAC+SLK (65±7.6 min) at a final concentration of 0.025 μM (APAC+SLK vs APAC, p<0.01). In comparison with APAC alone, the T50% of APAC+SLK was shortened by 1.56, 1.67 and 2.1 fold at the concentrations of 0.025, 0.5 and 0.1μM, respectively. Thus these low concentrations of a combination of both agents are likely to be more effective and less toxic when used therapeutically in vivo. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Decreased platelet reactivity in patients with cancer is associated with high risk of venous thromboembolism and poor prognosis

2017 ◽  
Vol 117 (01) ◽  
pp. 90-98 ◽  
Author(s):  
Julia Riedl ◽  
Alexandra Kaider ◽  
Christine Marosi ◽  
Gerald W. Prager ◽  
Beate Eichelberger ◽  
...  
Keyword(s):  
Venous Thromboembolism ◽  
Cancer Patients ◽  
Cancer Progression ◽  
Poor Prognosis ◽  
Ex Vivo ◽  
Platelet Reactivity ◽  
Platelet Response ◽  
Platelet Surface ◽  
Risk Of Death

SummaryPlatelets are suggested to play a crucial role in cancer progression and the prothrombotic state of cancer patients. Here, we aimed to examine the activation status of platelets in cancer patients and investigate their association with risk of death and occurrence of venous thromboembolism (VTE) in a prospective observational cohort study. We measured platelet surface P-selectin, activated glycoprotein (GP) IIb/IIIa and monocyte-platelet aggregate (MPA) formation in vivo and platelet response to ex vivo stimulation with agonists of protease-activated receptor (PAR) −1, −4, and GPVI, by whole blood flow cytometry, before beginning of chemotherapy and repeatedly during the first six months thereafter (total number of samples analysed: 230). Endpoints of the study were occurrence of death or VTE during a two-year follow-up, respectively. Of 62 patients (median age [interquartile range, IQR]: 63 [54–70] years, 48 % female), 32 (51.6 %) died and nine (14.5 %) developed VTE. Association with a higher risk of death was found for lower platelet surface expression of P-selectin and activated GPIIb/IIIa in vivo and in response to PAR-1, −4 and GPVI activation, but not for MPA formation. Furthermore, reduced platelet responsiveness to PAR-1 and GPVI agonists was associated with higher risk of VTE (hazard ratio per decile increase of percentage P-selectin positive platelets: 0.73 [0.56–0.92, p=0.007] and 0.77 [0.59–0.98, p=0.034], respectively). In conclusion, cancer patients with a poor prognosis showed decreased platelet reactivity, presumably as a consequence of continuous activation. Our data suggest that decreased platelet reactivity is associated with increased mortality and VTE in cancer.Supplementary Material to this article is available online at www.thrombosis-online.com.

scholarly journals In vivo effects of eltrombopag on platelet function in immune thrombocytopenia: no evidence of platelet activation

Blood ◽  
2012 ◽  
Vol 119 (17) ◽  
pp. 4066-4072 ◽  
Author(s):  
Bethan Psaila ◽  
James B. Bussel ◽  
Matthew D. Linden ◽  
Bracken Babula ◽  
Youfu Li ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Platelet Function ◽  
Whole Blood ◽  
Immune Thrombocytopenia ◽  
Ex Vivo ◽  
Platelet Reactivity ◽  
Adenosine Diphosphate ◽  
High Dose ◽  
Platelet Surface

Abstract The effects of eltrombopag, a thrombopoietin-receptor agonist, on platelet function in immune thrombocytopenia (ITP) are not fully characterized. This study used whole blood flow cytometry to examine platelet function in 20 patients receiving eltrombopag treatment at days 0, 7, and 28. Platelet surface expression of activated GPIIb/IIIa, P-selectin, and GPIb was measured with and without low and high adenosine diphosphate (ADP) and thrombin receptor activating peptide (TRAP) concentrations. Before eltrombopag treatment with no ex vivo agonist, platelet activation was higher in ITP patients than controls. Platelet GPIb and activated GPIIb/IIIa expression without added agonist was unchanged following eltrombopag treatment, whereas a slight increase in P-selectin was observed. Expression of P-selectin and activated GPIIb/IIIa in response to high-dose ADP was lower during eltrombopag treatment than at baseline. Eltrombopag led to a slight increase in platelet reactivity to TRAP only in responders to eltrombopag but not to levels above those in controls; whole blood experiments demonstrated that this increase was probably because of higher platelet counts rather than higher platelet reactivity. In conclusion, although thrombocytopenic ITP patients have higher baseline platelet activation than controls, eltrombopag did not cause platelet activation or hyper-reactivity, irrespective of whether the platelet count increased.

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