Two distinct pathways regulate platelet phosphatidylserine exposure and procoagulant function

Abstract Procoagulant platelets exhibit hallmark features of apoptotic cells, including membrane blebbing, microvesiculation, and phosphatidylserine (PS) exposure. Although platelets possess many well-known apoptotic regulators, their role in regulating the procoagulant function of platelets is unclear. To clarify this, we investigated the consequence of removing the essential mediators of apoptosis, Bak and Bax, or directly inducing apoptosis with the BH3 mimetic compound ABT-737. Treatment of platelets with ABT-737 triggered PS exposure and a marked increase in thrombin generation in vitro. This increase in procoagulant function was Bak/Bax- and caspase-dependent, but it was unaffected by inhibitors of platelet activation or by chelating extracellular calcium. In contrast, agonist-induced platelet procoagulant function was unchanged in Bak−/−Bax−/− or caspase inhibitor–treated platelets, but it was completely eliminated by extracellular calcium chelators or inhibitors of platelet activation. These studies show the existence of 2 distinct pathways regulating the procoagulant function of platelets.

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Role of HMGB1 in apoptosis-mediated sepsis lethality

Journal of Experimental Medicine ◽

10.1084/jem.20052203 ◽

2006 ◽

Vol 203 (7) ◽

pp. 1637-1642 ◽

Cited By ~ 256

Author(s):

Shixin Qin ◽

Haichao Wang ◽

Renqi Yuan ◽

Hui Li ◽

Mahendar Ochani ◽

...

Keyword(s):

Severe Sepsis ◽

High Mobility ◽

Organ Damage ◽

The United States ◽

Apoptotic Cells ◽

Caspase Inhibitor ◽

Common Pathway ◽

The Third

Severe sepsis, a lethal syndrome after infection or injury, is the third leading cause of mortality in the United States. The pathogenesis of severe sepsis is characterized by organ damage and accumulation of apoptotic lymphocytes in the spleen, thymus, and other organs. To examine the potential causal relationships of apoptosis to organ damage, we administered Z-VAD-FMK, a broad-spectrum caspase inhibitor, to mice with sepsis. We found that Z-VAD-FMK–treated septic mice had decreased levels of high mobility group box 1 (HMGB1), a critical cytokine mediator of organ damage in severe sepsis, and suppressed apoptosis in the spleen and thymus. In vitro, apoptotic cells activate macrophages to release HMGB1. Monoclonal antibodies against HMGB1 conferred protection against organ damage but did not prevent the accumulation of apoptotic cells in the spleen. Thus, our data indicate that HMGB1 production is downstream of apoptosis on the final common pathway to organ damage in severe sepsis.

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Injection of recombinant activated factor VII can induce transient increase in circulating procoagulant microparticles

Thrombosis and Haemostasis ◽

10.1160/th03-05-0301 ◽

2004 ◽

Vol 91 (05) ◽

pp. 873-878 ◽

Cited By ~ 21

Author(s):

Bénédicte Hugel ◽

Benoit Guillet ◽

Catherine Trichet ◽

Anne Rafowicz ◽

Thierry Lambert ◽

...

Keyword(s):

Short Duration ◽

Platelet Activation ◽

Thrombin Generation ◽

Transient Increase ◽

Factor Vii ◽

Recombinant Activated Factor Vii ◽

Activated Factor Vii ◽

Therapeutic Doses

SummaryRecombinant activated factor VII (rFVIIa) is an effective haemostatic treatment in haemophiliacs with inhibitors. In vitro, FVIIa concentrations corresponding to those obtained with therapeutic doses of rFVIIa have been shown to induce normal thrombin generation and platelet activation in the absence of factors VIII or IX. To further study the in vivo haemostatic changes induced by rFVIIa, circulating procoagulant microparticles (MP) were measured in patients treated with discontinuous injections of Novoseven®. In 6 out of 15 patients, a transient peak of procoagulant MP was observed after injection, occurring 15 min to 2 h after infusion. It was composed primarily of platelet-derived MP and was of very short duration. This peak was not observed in haemophiliacs without inhibitor, who were treated with conventional replacement therapies. Our results provide further in vivo evidence that rFVIIa specifically activates platelets, either directly or as a consequence of a burst of thrombin generation that could account for its haemostatic efficacy.

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Hemoperfusion leads to impairment in hemostasis and coagulation process in patients with acute pesticide intoxication

Scientific Reports ◽

10.1038/s41598-019-49738-1 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Samel Park ◽

Md-Imtiazul Islam ◽

Ji-Hun Jeong ◽

Nam-Jun Cho ◽

Ho-yeon Song ◽

...

Keyword(s):

Platelet Activation ◽

Thrombin Generation ◽

Degradation Products ◽

Treatment Modalities ◽

Platelet Surface ◽

Distribution Width ◽

Risk Of Bleeding ◽

Increased Risk ◽

Significant Change

Abstract Hemoperfusion (HP) is one of the important treatment modalities in extracorporeal therapy for patients with acute intoxication. Its use has declined during the past 20 years despite its efficacy, because of its side effects, especially an increased risk of bleeding. Mechanisms of hemostasis impairment have not been clearly elucidated and studies demonstrating the mechanism are lacking. It is not clear which step of the hemostatic process is impaired during HP, and whether it leads to an increased risk of bleeding. We performed both in vivo and in vitro studies to elucidate the mechanism of impairment in the hemostatic process. In patients with acute pesticide intoxication who underwent HP, the platelet count decreased rapidly during the first 30 minutes from 242.4 ± 57.7 × 103/μL to 184.8 ± 49.6 × 103/μL, then gradually decreased even lower to 145.4 ± 61.2 × 103/μL over time (p < 0.001). As markers of platelet activation, platelet distribution width increased continuously during HP from 41.98 ± 9.28% to 47.69 ± 11.18% (p < 0.05), however, mean platelet volume did not show significant change. In scanning electron microscopy, activated platelets adhered to modified charcoal were observed, and delayed closure time after HP in PFA-100 test suggested platelet dysfunction occurred during HP. To confirm these conflicting results, changes of glycoprotein expression on the platelet surface were evaluated when platelets were exposed to modified charcoal in vitro. Platelet expression of CD61, fibrinogen receptor, significantly decreased from 95.2 ± 0.9% to 73.9 ± 1.6%, while those expressing CD42b, von Willebrand factor receptor, did not show significant change. However, platelet expression of CD49b, collagen receptor, significantly increased from 24.6 ± 0.7% to 51.9 ± 2.3%. Thrombin-antithrombin complex, a marker for thrombin generation, appeared to decrease, however, it was not statistically significant. Fibrin degradation products and d-dimers, markers for fibrinolysis, increased significantly during HP. Taken together, our data suggests that hemoperfusion leads to impairment of platelet aggregation with incomplete platelet activation, which was associated with reduced thrombin generation, accompanied by increased fibrinolysis.

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A Role for Bile Salt-Dependent Lipase in Platelet Activation and in Thrombus Formation in Vivo.

Blood ◽

10.1182/blood.v104.11.3526.3526 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3526-3526 ◽

Cited By ~ 1

Author(s):

Laurence Panicot-Dubois ◽

Christophe Dubois ◽

Barbara C. Furie ◽

Bruce Furie ◽

Dominique Lombardo

Keyword(s):

Bile Salt ◽

Platelet Activation ◽

Thrombus Formation ◽

Pancreatic Acinar Cells ◽

Cremaster Muscle ◽

Terminal Domain ◽

Laser Injury ◽

Phosphatidylserine Exposure

Abstract Bile Salt Dependent Lipase (BSDL) is an enzyme secreted by pancreatic acinar cells. BSDL, in the presence of primary bile salts, participates in the hydrolysis of dietary lipid esters in the duodenum lumen. This 105 kDa N and O-glycosylated protein has been detected in the plasma of normal subjects. Recent in vitro and in vivo studies demonstrated that pancreatic BSDL reaches the blood via transcytosis through enterocytes. Other studies showed that pancreatic human BSDL is captured by human umbilical vein endothelial cells and induces the proliferation of smooth muscle cells in vitro at BSDL concentrations found in blood, suggesting that this enzyme may play a role in hemostasis and thrombosis. However the specific role of circulating BSDL is unknown. The goal of this study was to determine the possible involvement of circulating BSDL in thrombus formation. We investigated the participation of circulating mouse BSDL in thrombus formation using widefield intravital microscopy in the cremaster muscle of living mice. Thrombi were formed following laser injury of the vessel wall of an arteriole in the cremaster muscle. Pancreatic mouse BSDL, a 74 kDa glycoprotein, was detected using several antibodies directed against either the whole human BSDL (pAbL64, pAbL32) or a peptide based on a sequence in the N-terminal domain of BSDL (Ser326-Thr350; pAbAntipeptide). Mouse and human BSDL share about 80% sequence homology, the main difference localized in the C-terminal domain, which is truncated to the mouse BSDL compared with the human enzyme. All the antibodies are able to specifically recognize the mouse pancreatic BSDL. Using antibodies pAbL64, pAbL32, or pAbAntipeptide we observed specific accumulation of circulating mouse BSDL into the growing thrombus. The circulating BSDL co-localized with platelets present in the thrombus. These results suggest that circulating BSDL is involved in thrombus formation in vivo. In order to determine if BSDL plays a role in platelet activation and aggregation, we performed in vitro studies on human washed platelets. BSDL increased both the amount of phosphatidylserine exposure on the surface of platelets and the activation of αIIbβ3 induced by thrombin. These results indicate that this enzyme can amplify the activation of platelets in vitro. While BSDL alone cannot induce the aggregation of platelets, this enzyme significantly increases the amount of platelet aggregation induced by SFLLRN peptide or thrombin. Altogether, these data suggeste that circulating BSDL participates in the thrombus formation after laser injury of the arterial wall and can amplify both the activation of platelets and the phosphatidylserine exposure, increasing the thrombotic response after vessel injury. This mechanism may be operative in the development of venous thromboembolic disease in pancreatic cancer.

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Ex vivo synergistic effects of apixaban with dual anti-platelet therapy (DAPT) in lowering platelet reactivity and thrombin generation (SEARCH)

European Heart Journal ◽

10.1093/ehjci/ehaa946.1546 ◽

2020 ◽

Vol 41 (Supplement_2) ◽

Author(s):

B Curry ◽

J Kotha ◽

L Miller ◽

M Dixon ◽

M.J Herr ◽

...

Keyword(s):

Platelet Activation ◽

Thrombin Generation ◽

Platelet Reactivity ◽

Synergistic Effects ◽

Prior History ◽

Blood Collection ◽

Funding Source ◽

Synergistic Activity ◽

Anti Platelet Therapy

Abstract Direct oral anticoagulants such as apixaban are increasingly being evaluated clinically for the secondary prevention of cardiovascular events; however, their effects on platelet function in combination with dual anti-platelet therapy (DAPT) have not been fully investigated. The purpose of this translational, in vitro study was to determine if apixaban via inhibition of thrombin generation exhibits synergistic activity with DAPT to reduce platelet reactivity. Consented subjects with a prior history (<12 mo) of ACS on DAPT regime with aspirin and clopidogrel (n=15; DAPT-C) or aspirin and ticagrelor (n=15; DAPT-T) were recruited, along with the age-matched healthy subjects as controls. Enrolled DAPT subjects had taken their prescribed regimen >7 days prior to blood collection. Platelet-rich plasma from TSC anticoagulated blood was prepared and treated in vitro with nothing, a carrier control or apixaban (40, 90 and 220 ng/mL). The range of 40 to 220 ng/mL brackets the expected apixaban exposure at steady state with all three approved regimens with the 40 ng/mL treatment corresponding to <5th percentile for the 2.5 mg bid dose, the 90 ng/mL corresponding to Cmax after the 2.5 mg bid or to Cmin after the 5 mg bid dose, and the 220 ng/mL corresponding to the Cmax after 10 mg bid dose. Platelet aggregation was measured by light transmission aggregometry (LTA) with tissue factor (TF) as agonist. Platelet p-selectin expression was measured by flow cytometry and thrombin generation was quantified. TF agonist was chosen to evaluate endogenous thrombin effects via Factor Xa activation (fXa). The CaCl2 concentration in the TF was titrated in the presence of peptide GPRP which minimized fibrin generation. The baseline maximal aggregation (MA) response was similar for both DAPT-T and DAPT-C (64%). Compared to DAPT alone, 90 and 220 ng/mL apixaban treatments decreased MA from 64% to 36% and 17% in the DAPT-T group and from 64% to 28% and 9% in the DAPT-C group (p<0.009), respectively. Platelet P-selectin expression decreased by 53% in the DAPT-T group with 220 ng/mL apixaban treatment (p<0.02) and in the DAPT-C group by 70% and 76% with 90 and 220 ng/mL apixaban treatment (p<0.004), respectively, compared to DAPT alone. Apixaban treatment (90 and 220 ng/mL) significantly increased thrombin generation lag time and time-to-peak results and significantly decreased peak thrombin in both DAPT groups (p<0.05). ACS patients on a DAPT regimen were susceptible to thrombin-mediated platelet activation via fXa. Apixaban treatment in vitro caused a larger reduction in thrombin-mediated platelet activation in the clopidogrel group compared to the ticagrelor group, consistent with ticagrelor having a more potent anti-platelet effect compared to clopidogrel. The in vitro addition of apixaban that corresponded to currently approved dosing regimens and at plasma drug levels routinely achieved demonstrated synergy with DAPT to reduce platelet reactivity and thrombin generation. Funding Acknowledgement Type of funding source: Private grant(s) and/or Sponsorship. Main funding source(s): Bristol-Myers Squibb

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IN VITRO STUDIES ON THE EFFECT OF ACTIVATED PROTEIN C ON PLATELET ACTIVATION AND THROMBIN GENERATION

Thrombosis Research ◽

10.1016/s0049-3848(97)00119-9 ◽

1997 ◽

Vol 87 (2) ◽

pp. 197-204 ◽

Cited By ~ 9

Author(s):

B Kaiser ◽

W Jeske ◽

D.H Hoppensteadt ◽

J.M Walenga ◽

W Drohan ◽

...

Keyword(s):

Platelet Activation ◽

Protein C ◽

Thrombin Generation ◽

Activated Protein C ◽

In Vitro Studies

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Preclinical safety and efficacy evaluation of the Pipeline Vantage Embolization Device with Shield Technology

Journal of NeuroInterventional Surgery ◽

10.1136/neurintsurg-2020-016043 ◽

2020 ◽

Vol 12 (10) ◽

pp. 981-986 ◽

Cited By ~ 2

Author(s):

Robert M Starke ◽

John Thompson ◽

Ariana Pagani ◽

Animesh Choubey ◽

John M Wainwright ◽

...

Keyword(s):

Platelet Activation ◽

Thrombin Generation ◽

Flow Diverter ◽

Loop Model ◽

Flow Loop ◽

Efficacy Evaluation ◽

Vessel Patency ◽

Aneurysm Occlusion

BackgroundThe Pipeline Vantage Embolization Device with Shield Technology is a next generation flow diverter developed to improve aneurysm occlusion and implant endothelialization in addition to lowering thrombogenicity. We report here the in vivo biocompatibility and in vitro hemocompatibility performance of the Pipeline Vantage Embolization Device with Shield Technology (Vantage) compared with the Pipeline Flex Embolization Device (Flex).MethodsBiocompatibility (via histology), aneurysm occlusion and vessel patency (via angiography), and endothelial coverage (via scanning electron microscopy (SEM)) for the Vantage and Flex devices were assessed in the rabbit elastase aneurysm model at 90 days (n=29) and 180 days (n=27). In vitro thrombogenicity for Flex and Vantage (n=16) was assessed using a human blood flow loop model at low heparin concentration (0.6 U/mL) with thrombin generation, platelet activation and thrombus visualization as outputs.ResultsRaymond Roy Occlusion Classification grade 1 was higher for Vantage (61%) compared with Flex (46%), but was not statistically significant (p>0.05). All branch vessels were patent. Histological measures for both devices were similar (p>0.05). Endothelial coverage of the implant was significantly better for Vantage compared with Flex (p<0.05). In vitro measurements of thrombin generation (thrombin-antithrombin complex (µg/mL): Vantage 0.49±0.45; Flex 10.57±9.84) and platelet activation (β-thromboglobulin (IU/µl): Vantage 0.41±0.19; Flex 4.14±2.38) were both statistically lower (p<0.05) for Vantage compared with Flex. High resolution microscopy showed less accumulation of thrombus on Vantage as compared with Flex.ConclusionVantage improved aneurysm occlusion and implant endothelialization and had significantly lower thrombogenicity as compared with Flex, while preserving the biocompatibility safety profile of Flex.

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PDI Inhibition Blocks Thrombin Generation in Humans By Interfering with Platelet Factor V Activation

Blood ◽

10.1182/blood.v128.22.2628.2628 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2628-2628

Author(s):

Jack D Stopa ◽

Donna S. Neuberg ◽

Maneka Puligandla ◽

Bruce Furie ◽

Robert C. Flaumenhaft ◽

...

Keyword(s):

Platelet Activation ◽

Disulfide Bond ◽

Thrombin Generation ◽

Thrombus Formation ◽

Factor V ◽

Dependent Manner ◽

Plasma Samples ◽

Platelet Factor

Abstract Protein disulfide isomerase (PDI) is a ubiquitously expressed oxidoreductase that serves an essential role in protein folding in the endoplasmic reticulum by reshuffling disulfide bonds within nascent proteins. PDI can be released from vascular cells, including platelets, and inhibition or platelet-specific deletion of PDI blocks thrombus formation in vivo. However, the specific function of PDI in thrombus formation is poorly understood. Unlike the role of proteases in blood coagulation, which have been studied in depth, little is known about PDI substrates in the vasculature. Several platelet and endothelial integrins have been identified as putative substrates for PDI, but whether coagulation factors are directly targeted by extracellular PDI has not been established. We now identify platelet factor V as a principal coagulation substrate of extracellular PDI. We developed an unbiased strategy to identify novel substrates of PDI in washed platelets using PDI variants capable of trapping substrates: FLAG-tagged PDI mutants modified by a substitution of arginine or proline for histidine (CGHC → CGRC; CGHC → CGPC) in the catalytic motif of both the a and a' domains. Whereas the AGHA-PDI variant which has no catalytic activity serves as a control. The CGRC-PDI variant co-precipitated with platelet factor V in a redox-sensitive manner while there was no platelet factor V detected with the AGHA-PDI variant, thus confirming that binding of PDI to platelet-derived factor V occurs through disulfide bond exchange. Platelet factor V associates with multimerin-1 through a disulfide bond. Trapping PDI mutants also bind to multimerin-1 in a reaction requiring disulfide bond exchange. To evaluate the effect of PDI inhibition on the activation of platelet factor V, washed platelets from healthy donors were stimulated with 0.1 U/mL of thrombin in the presence of varying concentrations of isoquercetin (0 to 50 µM), which has previously been shown to inhibit PDI function. We observed a dose-dependent reduction of factor Va following platelet activation despite the fact that isoquercetin did not inhibit platelet release of PF4 or block Xa or thrombin enzymatic activity directly. We next performed a clinical study designed to determine whether oral isoquercetin inhibits thrombin generation in human subjects via its ability to inhibit platelet Va generation. Plasma samples collected from healthy participants before and 4 hours after ingestion of 1000 mg of isoquercetin (N=17). In plasma samples, post-isoquercetin platelet-dependent thrombin generation decreased by 51% compared with pre-ingestion controls (P=0.0004). Furthermore, we observed an overall 26% reduction in FVa in non-FV depleted plasma (P<0.001), which corresponded with a 53% decrease in FVa generated from platelets (P<0.001). These data confirm a significant effect of PDI inhibition on the generation of FVa following platelet activation. Considering that isoquercetin reduces platelet FVa generation and similarly inhibits platelet-dependent thrombin generation in a PDI-dependent manner, we investigated whether the addition of FVa in vitro restored platelet-dependent thrombin generation. The pre-incubation of 7 µg/mL FVa prior to stimulation with low dose thrombin restored platelet-dependent thrombin generation to within 80% baseline of pre-treatment levels. We conclude that platelet factor V is an essential substrate in mediating PDI-dependent thrombin generation on platelets and propose that PDI cleaves a disulfide bond that links platelet factor V to multimerin-1, thereby releasing platelet factor V for activation and subsequent thrombin generation. Disclosures Zwicker: Quercegen Pharma: Research Funding.

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Serotonergic mechanisms enhance platelet-mediated thrombogenicity

Thrombosis and Haemostasis ◽

10.1160/th08-12-0810 ◽

2009 ◽

Vol 102 (09) ◽

pp. 511-519 ◽

Cited By ~ 35

Author(s):

Irene Lopez-Vilchez ◽

Maribel Diaz-Ricart ◽

Fulgencio Navalon ◽

Esther Gomez ◽

Cristobal Gasto ◽

...

Keyword(s):

Platelet Activation ◽

Whole Blood ◽

Thrombin Generation ◽

Platelet Rich Plasma ◽

Thrombus Formation ◽

Selective Inhibition ◽

Human Platelets ◽

Experimental Conditions ◽

Perfusion Studies

SummaryAlthough it is generally acknowledged that serotonin (5-HT) is a weak agonist for human platelets, recent information suggests an association between serotonergic mechanisms and cardiovascular risk. We investigated the action of 5-HT on adhesive, cohesive and procoagulant properties of human platelets. Impact of 5-HT on whole blood coagulation and thrombin generation was measured by modified thromboelastometry (TEM) and specific fluorogenic assays. We evaluated the effects of 5-HT on thrombus formation in an in-vitro model of thrombosis using human flowing blood. In platelet-rich plasma (PRP), 5-HT favoured the expression of CD62-P, and procoagulant molecules on platelet membranes. These effects were potentiated in the presence of Ca++ and/or ADP. Incubation with 5-HT accelerated clotting times and augmented clot strength in whole blood TEM, and enhanced thrombin generation in PRP. In perfusion studies, 5-HT significantly increased fibrin deposition at low shear (300s-1) and enhanced platelet thrombus formation on the damaged vascular surface at high shear (1,200s-1). Selective inhibition of serotonin reuptake (SSRI) attenuated effects of 5-HT on platelet activation and downregulated the prothrombotic tendencies observed in the previous experimental conditions. In general, reductions of thrombogenic patterns observed with SSRI were more evident under shear conditions (aggregation and perfusion systems) and less evident under steady conditions (TEM and thrombin generation assays). In conclusion, 5-HT is not a weak agonist for human platelets; instead it accentuates platelet activation, potentiates procoagulant responses on human blood and increases thrombogenesis on damaged vascular surfaces. The remarkable antithrombotic actions achieved through SSRI deserve further mechanistic and clinical investigations.

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

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615117 ◽

1998 ◽

Vol 79 (05) ◽

pp. 1041-1047 ◽

Cited By ~ 20

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.

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