Abnormal plasma clot structure and stability distinguish bleeding risk in patients with severe factor XI deficiency
2014; Elsevier BV; Volume: 12; Issue: 7 Linguagem: Inglês
10.1111/jth.12600
ISSN1538-7933
AutoresM. Zucker, U. Seligsohn, Ophira Salomon, Alisa S. Wolberg,
Tópico(s)Vitamin K Research Studies
ResumoJournal of Thrombosis and HaemostasisVolume 12, Issue 7 p. 1121-1130 Original ArticleFree Access Abnormal plasma clot structure and stability distinguish bleeding risk in patients with severe factor XI deficiency M. Zucker, Corresponding Author M. Zucker Thrombosis and Hemostasis Unit, Amalia Biron Research Institute of Thrombosis and Hemostasis, Sheba Medical Center, Tel Hashomer and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Correspondence: Michal Zucker, Amalia Biron Research Institute of Thrombosis and Hemostasis, Sheba Medical Center, Tel Hashomer, Ramat Gan, Israel. Tel.: +972 3 530 7350; fax: +972 3 535 1568. Alisa S. Wolberg, Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, 815 Brinkhous-Bullitt Building, CB #7525, Chapel Hill, NC 27599-7525, USA. Tel.: +919 966 8430; fax: +919 966 6718. E-mails: michal.zucker@sheba.health.gov.il; alisa_wolberg@med.unc.eduSearch for more papers by this authorU. Seligsohn, U. Seligsohn Thrombosis and Hemostasis Unit, Amalia Biron Research Institute of Thrombosis and Hemostasis, Sheba Medical Center, Tel Hashomer and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, IsraelSearch for more papers by this authorO. Salomon, O. Salomon Thrombosis and Hemostasis Unit, Amalia Biron Research Institute of Thrombosis and Hemostasis, Sheba Medical Center, Tel Hashomer and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, IsraelSearch for more papers by this authorA. S. Wolberg, Corresponding Author A. S. Wolberg Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Correspondence: Michal Zucker, Amalia Biron Research Institute of Thrombosis and Hemostasis, Sheba Medical Center, Tel Hashomer, Ramat Gan, Israel. Tel.: +972 3 530 7350; fax: +972 3 535 1568. Alisa S. Wolberg, Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, 815 Brinkhous-Bullitt Building, CB #7525, Chapel Hill, NC 27599-7525, USA. Tel.: +919 966 8430; fax: +919 966 6718. E-mails: michal.zucker@sheba.health.gov.il; alisa_wolberg@med.unc.eduSearch for more papers by this author M. Zucker, Corresponding Author M. Zucker Thrombosis and Hemostasis Unit, Amalia Biron Research Institute of Thrombosis and Hemostasis, Sheba Medical Center, Tel Hashomer and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Correspondence: Michal Zucker, Amalia Biron Research Institute of Thrombosis and Hemostasis, Sheba Medical Center, Tel Hashomer, Ramat Gan, Israel. Tel.: +972 3 530 7350; fax: +972 3 535 1568. Alisa S. Wolberg, Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, 815 Brinkhous-Bullitt Building, CB #7525, Chapel Hill, NC 27599-7525, USA. Tel.: +919 966 8430; fax: +919 966 6718. E-mails: michal.zucker@sheba.health.gov.il; alisa_wolberg@med.unc.eduSearch for more papers by this authorU. Seligsohn, U. Seligsohn Thrombosis and Hemostasis Unit, Amalia Biron Research Institute of Thrombosis and Hemostasis, Sheba Medical Center, Tel Hashomer and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, IsraelSearch for more papers by this authorO. Salomon, O. Salomon Thrombosis and Hemostasis Unit, Amalia Biron Research Institute of Thrombosis and Hemostasis, Sheba Medical Center, Tel Hashomer and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, IsraelSearch for more papers by this authorA. S. Wolberg, Corresponding Author A. S. Wolberg Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Correspondence: Michal Zucker, Amalia Biron Research Institute of Thrombosis and Hemostasis, Sheba Medical Center, Tel Hashomer, Ramat Gan, Israel. Tel.: +972 3 530 7350; fax: +972 3 535 1568. Alisa S. Wolberg, Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, 815 Brinkhous-Bullitt Building, CB #7525, Chapel Hill, NC 27599-7525, USA. Tel.: +919 966 8430; fax: +919 966 6718. E-mails: michal.zucker@sheba.health.gov.il; alisa_wolberg@med.unc.eduSearch for more papers by this author First published: 11 May 2014 https://doi.org/10.1111/jth.12600Citations: 61 Manuscript handled by: T. Lisman Final decision: P. H. Reitsma, 25 April 2014 AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Summary Background Factor XI (FXI) deficiency is a rare autosomal recessive disorder. Many patients with even very low FXI levels (< 20 IU dL−1) are asymptomatic or exhibit only mild bleeding, whereas others experience severe bleeding, usually following trauma. Neither FXI antigen nor activity predicts the risk of bleeding in FXI-deficient patients. Objectives (i) Characterize the formation, structure and stability of plasma clots from patients with severe FXI deficiency and (ii) determine whether these assays can distinguish asymptomatic patients (‘non-bleeders’) from those with a history of bleeding (‘bleeders’). Methods Platelet-poor plasmas were prepared from 16 severe FXI-deficient patients who were divided into bleeders or non-bleeders, based on bleeding associated with at least two tooth extractions without prophylaxis. Clot formation was triggered by recalcification and addition of tissue factor and phospholipids in the absence or presence of tissue plasminogen activator and/or thrombomodulin. Clot formation and fibrinolysis were measured by turbidity and fibrin network structure by laser scanning confocal microscopy. Results Non-bleeders and bleeders had similarly low FXI levels, normal prothrombin times, normal levels of fibrinogen, factor VIII, von Willebrand factor and factor XIII, and normal platelet number and function. Compared with non-bleeders, bleeders exhibited lower fibrin network density and lower clot stability in the presence of tissue plasminogen activator. In the presence of thrombomodulin, seven of eight bleeders failed to form a clot, whereas only three of eight non-bleeders did not clot. Conclusions Plasma clot structure and stability assays distinguished non-bleeders from bleeders. These assays may reveal hemostatic mechanisms in FXI-deficient patients and have clinical utility for assessing the risk of bleeding. Introduction Factor XI (FXI) deficiency is a rare, autosomal recessive disorder present in 1 : 1 000 000 individuals. Homozygous or compound heterozygous patients have low FXI levels (< 20 IU dL−1), whereas heterozygotes have moderately reduced FXI levels (25–70 IU dL−1) 1, 2. Spontaneous bleeding is rare; however, patients with severe FXI deficiency can present with tissue-specific bleeding following surgery or injury, predominantly at sites with high fibrinolytic activity (mouth, nose and genitourinary tract), or menorrhagia 1, 2. Previous studies have shown that when clotting is initiated with low tissue factor (TF) concentrations, the FXI level mediates thrombin generation, fibrin formation and inhibition of fibrinolysis. Reduced FXI levels lead to diminished thrombin generation and reduced rate of fibrin formation 3-6. Subsequent studies extended these observations and showed that reduced thrombin generation results in reduced activation of the thrombin-activatable fibrinolysis inhibitor (TAFI) 7, 8 and consequently, reduced resistance of clots to fibrinolysis. These findings suggest an essential role for FXI in normal blood coagulation and clot stability, and provide a rationale for increased risk of bleeding in FXI-deficient patients. Interestingly, however, patients with similarly reduced FXI antigen and activity levels exhibit variable bleeding tendencies 9-11. Some patients are asymptomatic even after trauma, while others display bleeding with trauma, or bleeding that begins several hours or even days following trauma. Neither FXI antigen nor activity correlate with clinical risk of bleeding, and activated partial thromboplastin time (APTT) assays do not predict bleeding risk. Attempts to differentiate FXI-deficient patients with or without a bleeding tendency have focused on plasma thrombin generation characteristics. Rugeri et al. 12 isolated contact-inhibited platelet-rich plasma from healthy controls and patients with FXI deficiency divided into severe and mild/non-bleeder groups and measured thrombin generation triggered by addition of low TF. They showed that compared with controls, mild/non-bleeders have normal thrombin generation, but severe bleeders exhibit prolonged lag times and reduced rates and peaks of thrombin generation. In contrast, Guéguen et al. 13 did not detect significant differences in the thrombin generation peaks or endogenous thrombin potentials of either platelet-rich or platelet-poor plasma isolated from FXI-deficient patients categorized as bleeders or non-bleeders, although lag times in platelet-rich plasmas from bleeders were slightly (non-significantly, P = 0.07) prolonged compared with those from non-bleeders. Neither of these studies evaluated plasma clot formation or stability. Consequently, the relationship between plasma clot quality and bleeding risk in these patients remains unknown. Herein, we characterize plasma clot formation and quality in severe FXI-deficient patients and show that plasma clotting assays can identify patients with increased risk of bleeding. Materials and methods Materials Corn trypsin inhibitor and rabbit lung thrombomodulin were from Haematologic Technologies, Inc. (Essex Junction, VT, USA). Innovin (human TF) was from Siemens Healthcare Diagnostics (Newark, DE, USA). Tissue plasminogen activator (t-PA) was from American Diagnostica, Inc. (Stamford, CT, USA). AlexaFluor488-conjugated fibrinogen (6 mol dye per mol fibrinogen) was prepared as previously described 14. Human subjects The FXI-deficient cohort consisted of 16 unrelated Israeli patients who were referred to the Sheba Medical Center for evaluation of a bleeding tendency or prolonged APTT, and whose FXI level was < 9 IU dL−1. A subgroup of eight patients (five males, three females) was defined as ‘bleeders’ because they previously bled excessively following at least two separate sessions of tooth extractions performed with no prophylaxis. Excessive bleeding was determined when patients had oozing of blood for 1 h or more after extraction, when bleeding reoccurred within 24 h, or when the patient returned to the clinic or was hospitalized to achieve hemostasis. ‘Non-bleeders’ were eight patients (four male and four female) who underwent at least two uneventful tooth extractions without prophylaxis (Table 1). Bleeding histories were obtained by two experienced clinicians and patients were classified as bleeders or non-bleeders prior to further laboratory testing. A cohort of healthy controls consisted of 10 unrelated individuals (four male and six female) with normal levels of FXI. The mean age of patients (bleeders and non-bleeders) was 57 ± 15 years and of controls was 51 ± 11 years (Table 1). Table 1. Demographic characteristics of subjects and clinical coagulation tests and clotting factor levels Controls (n = 10) Non-bleeders (n = 8) Bleeders (n = 8) P anova P* NA, not assayed. Mean ± SD. *anova with Bonferroni post-hoc test. †t-test. ‡FXIII:Ag only for two controls. Bold values indicate significant P values. Controls vs. non-bleeders P* NA, not assayed. Mean ± SD. *anova with Bonferroni post-hoc test. †t-test. ‡FXIII:Ag only for two controls. Bold values indicate significant P values. Controls vs. bleeders P* NA, not assayed. Mean ± SD. *anova with Bonferroni post-hoc test. †t-test. ‡FXIII:Ag only for two controls. Bold values indicate significant P values. Bleeders vs. non-bleeders Age (years, mean ± SD) 51 ± 11 58 ± 7 57 ± 21 0.41 – – – Male, n (%) 4 (40) 4 (50) 5 (63) 0.67 – – – Prothrombin time (%) 107 ± 7 105 ± 13 104 ± 16 0.88 – – – APTT (s) 31 ± 3 56 ± 6 68 ± 24 < 0.0001 < 0.01 < 0.001 0.11 Thrombin time (s) 14.3 ± 1.6 14.9 ± 1.3 14.6 ± 0.5 0.65 – – – XI:Ac (%) 114 ± 21.0 3 ± 1.4 3.34 ± 3.0 < 0.0001 < 0.001 < 0.001 0.40 Fibrinogen (mg dL−1) 310 ± 51 318 ± 51 310 ± 35 0.93 – – – VIII:Ac (%) 96 ± 27 104 ± 27 108 ± 24 0.63 – – – VWF:Ac (%) 127 ± 38 161 ± 48 152 ± 49 0.27 – – – XIII:Ag (%) 108 ± 13‡ NA, not assayed. Mean ± SD. *anova with Bonferroni post-hoc test. †t-test. ‡FXIII:Ag only for two controls. Bold values indicate significant P values. 116 ± 20 107 ± 25 0.60 – – – Antithrombin (%) 93 ± 22 107 ± 17 102 ± 17 0.23 – – – TFPI (ng mL−1) 45.0 ± 19.8 54.0 ± 16.4 72.2 ± 32.8 0.07 – – – Protein C (%) 103 ± 16 112 ± 18 107 ± 32 0.68 – – – Protein S:Ac (%) 94 ± 18 100 ± 17 113 ± 28 0.20 – – – TAFI:Ag (%) 62 ± 18 78 ± 31 87 ± 35 0.59 – – – PAI-1 (U mL−1) NA 4.2 ± 3.4 4.2 ± 2.8 0.50a NA, not assayed. Mean ± SD. *anova with Bonferroni post-hoc test. †t-test. ‡FXIII:Ag only for two controls. Bold values indicate significant P values. – – – Platelets, μL−1 219 700 ± 46 162 207 000 ± 71 893 217 000 ± 34 297 0.87 – – – Platelet aggregation (%, collagen) 88.0 ± 3 90.7 ± 3 89.5 ± 3 0.13 – – – Platelet aggregation (%, ADP) 85.6 ± 6 90.4 ± 6 84.5 ± 5 0.11 – – – Platelet aggregation (%, epinephrine) 85 ± 3 79 ± 20 77 ± 20 0.50 – – – NA, not assayed. Mean ± SD. *anova with Bonferroni post-hoc test. †t-test. ‡FXIII:Ag only for two controls. Bold values indicate significant P values. Plasma preparation Informed consent was obtained from each donor in accordance with the Declaration of Helsinki. Blood was collected by venipuncture through a 21-gauge, butterfly needle into a syringe via a protocol approved by the Institutional Review Board of the Sheba Medical Center. The first 5 mL were discarded. The following 30 mL were drawn into a separate syringe containing sodium citrate/corn trypsin inhibitor (0.109 M/3.2% sodium citrate, pH 6.5, 18.3 μg mL−1 corn trypsin inhibitor) to minimize contact activation 15. Platelet-poor plasma (PPP) was prepared by sequential centrifugation (150 × g for 15 min, 1500 × g for 15 min), aliquoted and snap-frozen in −20 °C within 2 h of blood collection. All analyses of plasma thrombin generation and clot formation, structure and stability were performed in a blinded fashion. Clinical coagulation testing Prothrombin time, APTT, thrombin time, fibrinogen levels, plasma FXI and FVIII activity levels were measured by ACL-TOP-500 (Instrumental Laboratories, Bedford, MA, USA), using RecombiPlasTin 2G, SynthASil, ThrombinTime, Fibrinogen C reagents, FXI-deficient plasma and FVIII-deficient plasma, respectively (HemosIL; Beckman Coulter Inc., Nyon, Switzerland). Von Willebrand factor (VWF) and protein S antigen levels were measured by ACL-TOP-500, using the von Willebrand antigen kit and free protein S antigen (HemosiL). PAI-1 activity levels were measured by Sysmex 1500 using the Berichrom PAI kit (Siemens Healthcare Diagnostics, Marburg, Germany). Factor XIII, antithrombin and protein C activities were measured by chromogenic assays using Berichrom FXIII reagent, liquid antithrombin and the Coamatic protein C chromagenix kit (Siemens). Platelet aggregation was evaluated by light transmission aggregometry (AggRAM; Helena Laboratories, Beaumont, TX, USA) using adenosine diphosphate (ADP, 10 μm; Diamed AG, Cressier, Switzerland), epinephrine (50 μm; Diamed AG) or collagen (9 μm mL−1; Helena Laboratories) as platelet agonists. Changes in light transmission were recorded for 5 min and the aggregation maximal amplitude was measured. TAFI and tissue factor pathway inhibitor (TFPI) antigen levels were measured by ELISA (IMUCLONE TAFI ELISA kit, American Diagnostica Inc., and Human TFPI ELISA, RayBiotech Inc., Norcross, GA, USA). Phospholipid vesicles Phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine were from Avanti Polar Lipids (Alabaster, AL, USA). Large unilamellar vesicles (41% phosphatidylcholine/44% phosphatidylethanolamine/15% phosphatidylserine) were made as previously described 16. Briefly, lipids were combined, dried under nitrogen gas and resuspended in cyclohexanes. Resuspended lipids were lyophilized, resuspended in 20 mm N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) pH 7.4, 150 mm NaCl containing 1 mm ethylenediamine tetraacetic acid, and extruded through a 0.2 μm filter 10 times. Thrombin generation assays Thrombin generation was measured by calibrated automated thrombography 17. Briefly, TF/phospholipids were mixed with plasma in a 96-well round-bottom microtiter plate (Falcon™; Becton Dickinson, Franklin Lakes, NJ, USA), inserted into a Fluoroskan Ascent® fluorometer (ThermoLabsystem, Helsinki, Finland), and warmed to 37 °C for 10 min. Reactions were initiated by automatically dispensing fluorogenic substrate and CaCl2 to each well. Final TF, phospholipid, fluorogenic substrate and CaCl2 concentrations were 1 pm, 4 μm, 416 μm and 16 mm, respectively. Thrombin parameters (lag time, time to peak, peak and endogenous thrombin potential [ETP]) were calculated using Thrombinoscope software version 3.0.0.29 (Thrombinoscope BV, Maastricht, Netherlands), as we have described 18. Characterization of clot formation and lysis Clotting was initiated by incubating recalcified (10 mm CaCl2, final) PPP with TF and phospholipids (1 : 30 000 dilution of Innovin [0.5 pm TF] and 4 μm, final, respectively) in the absence or presence of t-PA (0.5 μg mL−1, final) and thrombomodulin (5 nm, final). Final reaction volumes were 100 μL (90% and 85% PPP, final, for clotting and fibrinolysis assays, respectively) in 96-well plates. Clot formation and lysis were monitored by turbidity at 405 nm in a SpectraMax 340Plus plate reader (Molecular Devices, Sunnyvale, CA, USA) for 2 h at room temperature. The onset of clot formation was the time to the inflection point prior to the turbidity increase. The maximum slope was the slope of a line fitted to the maximum rate of turbidity increase (‘Vmax’) using 5–10 points to determine the line. The peak turbidity change was the maximum turbidity of the clot less the starting turbidity of the plasma sample. For lysis experiments, the time to peak turbidity was the time to the inflection point at peak turbidity, and the area under the curve was the sum of trapezoids formed by turbidity curves less a baseline established by the lowest measurement recorded (Softmax Pro 5.4; Molecular Devices). Fibrin structure analysis Clots were produced by incubating recalcified (10 mm CaCl2, final) PPP with TF and phospholipids (1 : 30 000 dilution of Innovin [0.5 pm TF] and 4 μm, final, respectively) in Labtek II glass chamber slides. Plasmas were spiked with trace AlexaFluor488-conjugated fibrinogen (80 μg mL−1, final, 2.6% of total fibrinogen) to visualize fibrin fibers, as previously described 14. Clots were scanned with a Zeiss LSM700 confocal laser scanning microscope (Carl Zeiss, Inc., Thornwood, NY, USA) linked to a Zeiss inverted microscope equipped with a Zeiss 63× oil immersion plan apo-chromatic lens, as previously described 14. Thirty optical sections (1024 × 1024 pixels) were collected at 0.36-μm intervals in the z-axis. Images were processed using 3D deconvolution algorithms in AutoQuant software X 3.0.1 (Media Cybernetics Inc., Bethesda, MD, USA) prior to image analysis. Fibrin density was determined using ImageJ 1.41o by summing individual sections to create Z-projections, as described 19. Briefly, thresholding was performed on Z-projections using the ImageJ thresholding function to visualize fibers and minimize noise. Thresholds were set on a per experiment basis, to compensate for differences in gain settings. The area covered by pixels above the threshold cut-off was determined using the ImageJ Measure function. Because fiber diameter (~200–400 nm) is at the lower resolution limit of laser scanning confocal microscopy 20, 21, we did not quantify fibrin diameter. Statistical analyses All thrombin generation and clot formation, structure and fibrinolysis experiments were carried out in a blinded fashion. Descriptive statistics for clotting, fibrinolysis and fibrin structure parameters were summarized using means and standard deviations (SDs). Parameters were compared between groups using analysis of variance (anova) in Kaleidagraph version 4.1.3 (Synergy Software, Reading, PA, USA). Parameters showing significant differences were then analyzed with Bonferroni post hoc testing. P < 0.05 was considered significant. Results Coagulation tests and clotting factor levels do not correlate with bleeding risk in severe FXI-deficient patients Compared with healthy controls, both non-bleeders and bleeders had similarly reduced levels of FXI (3.0 ± 1.4 vs. 3.3 ± 3.0%, respectively) and prolonged APTTs (56 ± 6 and 68 ± 24 s, respectively). Both non-bleeders and bleeders had normal prothrombin and thrombin times and normal levels of fibrinogen, FVIII, VWF, factor XIII, antithrombin, proteins C and S and TAFI, and normal platelet numbers and function (Table 1). Bleeders had higher levels of TFPI than controls or non-bleeders, although these differences did not reach statistical significance (Table 1). Thrombin generation parameters do not differ between FXI-deficient patients and controls We first measured thrombin generation in plasmas from control individuals and FXI-deficient patients using calibrated automated thrombography. Although both non-bleeders and bleeders showed slightly prolonged lag times and times to peak and decreased peak thrombin generation compared with controls, these differences did not reach significance (Table 2). There were no differences in thrombin generation parameters between non-bleeders and bleeders (Table 2). Table 2. Thrombin generation parameters Controls (n = 9) Non-bleeders (n = 8) Bleeders (n = 8) P anova P Controls vs. non-bleeders P Controls vs. bleeders P Bleeders vs. non-bleeders Lag time (min) 4.2 ± 1.2 5.1 ± 1.7 4.9 ± 1.0 0.37 – – – Time to peak (min) 9.5 ± 2.4 11.4 ± 2.9 10.8 ± 2.4 0.32 – – – Thrombin peak (nm) 86.1 ± 59.7 57.1 ± 26.3 57.6 ± 41.5 0.33 – – – ETP (nm min) 809 ± 387 789 ± 319 684 ± 394 0.76 – – – Mean ± SD. Reduced plasma clot formation rate reflects FXI deficiency We then triggered clotting in plasmas from control individuals and FXI-deficient patients by recalcification and addition of TF and phospholipids, and monitored clot formation by turbidity (Fig. 1A). The onset times of fibrin formation for both bleeder and non-bleeder clots were prolonged compared with those seen in control clots, although these differences did not reach statistical significance (Table 3). Compared with controls, both bleeders and non-bleeders had a significantly slower rate (Vmax) of clot formation (Fig. 1B, Table 3), suggesting the FXI level modulates the clot formation rate. Both bleeders and non-bleeders also had a prolonged time to turbidity plateau (maximal fibrin formation), though only bleeders were significantly different from controls (P < 0.003, Fig. 1C, Table 3). These data suggest that the FXI level is a major determinant of the clot formation rate. Table 3. Plasma clot formation and fibrinolysis parameters Controls (n = 10) Non-bleeders (n = 8) Bleeders (n = 8) P anova P* Mean ± SD. *anova with Bonferroni post-hoc test. †Length of time between the midpoints of the increase and decrease in turbidity. A.U., arbitrary units. Bold values indicate significant P values. Controls vs. non-bleeders P* Mean ± SD. *anova with Bonferroni post-hoc test. †Length of time between the midpoints of the increase and decrease in turbidity. A.U., arbitrary units. Bold values indicate significant P values. Controls vs. bleeders P* Mean ± SD. *anova with Bonferroni post-hoc test. †Length of time between the midpoints of the increase and decrease in turbidity. A.U., arbitrary units. Bold values indicate significant P values. Bleeders vs. non-bleeders Clotting onset time (min) 13.4 ± 7.8 16.0 ± 7.4 23.8 ± 16.9 0.16 – – – Vmax (mOD min−1) 143.4 ± 67.5 43.3 ± 16.9 32.4 ± 25.5 < 0.0001 0.0003 < 0.0001 1 Time to plateau (min) 28.4 ± 15.3 47.4 ± 17.4 74.5 ± 37.4 < 0.003 0.34 < 0.003 0.14 Peak turbidity change 0.69 ± 0.14 0.68 ± 0.13 0.56 ± 0.11 0.07 – – – Fibrin density (A.U.) 222 786 ± 37 171 234 574 ± 42 621 177 387 ± 22 610 < 0.008 1 < 0.05 < 0.02 Fibrinolysis Onset time (min) 15.8 ± 8.8 11.6 ± 4.1 17.0 ± 6.1 0.28 – – – Vmax (mOD min−1) 166.0 ± 50.0 49.5 ± 12.5 39.5 ± 16.6 < 0.0001 < 0.0001 < 0.0001 1 Time to peak (min) 27.0 ± 11.3 32.2 ± 7.4 43.2 ± 17.3 < 0.05 1 < 0.05 0.28 Lysis timea NA, not assayed. Mean ± SD. *anova with Bonferroni post-hoc test. †t-test. ‡FXIII:Ag only for two controls. Bold values indicate significant P values. (min) 36.3 ± 11.4 29.9 ± 5.6 23.7 ± 3.5 < 0.02 0.32 < 0.01 0.39 Peak turbidity change 0.64 ± 0.11 0.58 ± 0.14 0.40 ± 0.12 < 0.002 0.83 < 0.002 < 0.05 Area under the curve 1482 ± 530 1176 ± 386 673 ± 210 < 0.002 0.39 < 0.002 <0.07 Mean ± SD. *anova with Bonferroni post-hoc test. †Length of time between the midpoints of the increase and decrease in turbidity. A.U., arbitrary units. Bold values indicate significant P values. Figure 1Open in figure viewerPowerPoint Plasma clot formation correlates with bleeding risk in FXI-deficient patients. Clotting was triggered in plasmas from healthy individuals and FXI-deficient patients in a blinded fashion by recalcification and addition of TF and phospholipids. Clot formation was monitored by turbidity. (A) Representative clot formation curves. (B) Rate (Vmax) of clot formation in controls and FXI-deficient patients (non-bleeders and bleeders, as indicated). (C) Time to plateau of turbidity in controls and FXI-deficient patients (non-bleeders and bleeders, as indicated). The boxes enclose 50% of the data with the median value displayed as a horizontal line, and lines enclose the interquartile distance (IQD). Open symbols represent points whose value falls more than 1.5-fold outside the IQD. Fibrin network structure correlates with bleeding risk in severe FXI-deficient patients Given the differences in the clot formation parameters between control and FXI-deficient plasmas, we measured the fibrin structure of clots produced from these plasmas. Plasmas were recalcified and clot formation was triggered by addition of TF and phospholipids. Fluorescently-labeled fibrinogen was included as a tracer in these reactions to visualize fibrin network structure by confocal microscopy, as previously described 14. Whereas fibrin structure in plasma clots from non-bleeders did not differ from controls, fibrin network density in plasma clots from bleeders was reduced ~20–25% compared with both controls (P < 0.05) and non-bleeders (P < 0.02) (Fig. 2A–B, Table 3). These findings demonstrate a unique feature of clots formed in bleeders. Specifically, FXI-deficient patients with increased bleeding produce abnormal fibrin network structure following TF-initiated clotting. Figure 2Open in figure viewerPowerPoint Fibrin network structure correlates with bleeding risk in FXI-deficient patients. Clots were formed in a blinded fashion by recalcification and addition of TF and phospholipids in the presence of AlexFluor488-conjugated fibrinogen. Laser scanning confocal microscopy was performed as described in 3. (A) Representative confocal micrographs (z-projections of 30 individual slices) of clots formed in plasma from control individuals and FXI-deficient patients (non-bleeders and bleeders, as indicated). (B) Fibrin network density (arbitrary units, A.U.) for controls and FXI-deficient patients (non-bleeders and bleeders, as indicated). The boxes enclose 50% of the data with the median value displayed as a horizontal line, and lines enclose the IQD. Open symbols represent points whose value falls more than 1.5-fold outside the IQD. Plasma clot stability correlates with bleeding risk in severe FXI-deficient patients The fibrin formation rate and fibrin network structure are major determinants of the ability of clots to withstand fibrinolysis; both reduced clot formation rate and reduced fibrin density are associated with increased susceptibility to fibrinolysis 19, 22. To determine the ability of control and FXI-deficient plasma clots to resist fibrinolysis, clotting reactions were initiated by recalcification and addition of TF and phospholipids in the presence of t-PA. In this assay, clot formation competes with clot lysis, which is detected as an increase and subsequent decrease in turbidity (Fig. 3A) 23-25. Similar to that seen in the absence of t-PA, bo
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