Original article 533
Effect of von Willebrand factor on clot structure and lysis Rita Marchia and He´ctor Rojasb Von Willebrand Factor (vWF) is constitutively secreted by the endothelium and incorporated in the fibrin clots under slow clotting conditions. The aim of the present work was to study the effect of vWF on clot structure and lysis. Purified fibrinogen was mixed with vWF or Tris-buffered saline and clotted with thrombin – activated factor XIII. Fibrin polymerization was followed by turbidity at 350 nm during 2.5 h. After this time, plasmin was added on the top of the clots, and the optical density (OD) was read until baseline values. vWF effect on network´s porosity was evaluated by permeation using the same clotting conditions as for fibrin polymerization. Clot structure was visualized and analyzed by laser scanning confocal microscopy (LSCM). The rate of fibrin polymerization was 1.47 mOD/s in the presence of vWF and 0.5 mOD/s when vWF was not added (P < 0.05). The fibrin lysis rate was approximately four times faster when vWF was added to fibrinogen. The fibrin network porosity was (20.4 W 1.6) T 10S9 cm2 with vWF and (8.3 W 1.2) T 10S9 cm2 without external vWF (P < 0.05). The analysis of LSCM images showed that vWF increased fibrin fibers diameter and the networks´ pores size. In conclusion,
Introduction Von Willebrand factor (vWF) is a large, multimeric plasma glycoprotein that plays an essential role in the initial adhesion of platelets to subendothelial connective tissue at the sites of vascular injury. vWF also is a carrier protein of blood clotting factor VIII (FVIII), protecting it from proteolytic degradation [1], and is assembled from identical subunits of approximately 250 kDa, which range in size from dimers of 500 kDa to multimers more than 20 000 kDa. The functional and/or quantitative vWF abnormalities cause the von Willebrand factor disease (vWFD), which is the most common inherited bleeding disorder [2]. The structure of the clot can be characterized by the fiber diameter, density, number and nature of the branch points (tri or tetramolecular), distances between branch points and size of the pores [3]. A variety of proteins bind to the fibrin clot through noncovalent and/or covalent bonds modifying its structure and the rate of fibrin degradation [4,5]. Actin, fibronectin, vWF and complement C3 [6], and the fibrinolytic inhibitors a2-antiplasmin, plasminogen activator inhibitor 2 (PAI 2) or thrombin activatable fibrinolysis inhibitor (TAFI), are crosslinked to fibrin by activated factor XIII (FXIIIa) [7]. FXIII is a transglutaminase, which catalyzes the formation of covalent e-(g-glutamyl)lysine crosslinks between the g-carboxy-amine group of a glutamine (amine acceptor) and the e-amino group of a lysine 0957-5235 Copyright ß 2015 Wolters Kluwer Health, Inc. All rights reserved.
vWF covalently crosslinked to fibrin modify its structure (increases fibrin diameter and the pores filling space of the meshwork) that accelerates the fibrin lysis rate. Blood Coagul Fibrinolysis 26:533–536 Copyright ß 2015 Wolters Kluwer Health, Inc. All rights reserved.
Blood Coagulation and Fibrinolysis 2015, 26:533–536 Keywords: confocal microscopy, fibrin polymerization, fibrinolysis, permeation, von Willebrand factor a Lab. Biologı´a del Desarrollo de la Hemostasia, IVIC and bInstituto de Inmunologı´a, Universidad Central de Venezuela (UCV), Caracas, Repu´blica Bolivariana de Venezuela
Correspondence to Rita Marchi Cappelletti, Centro de Medicina Experimental, Laboratorio de Fisiopatologı´a, Seccio´n Biologı´a del Desarrollo de la Hemostasia; Instituto Venezolano de Investigaciones, Cientı´ficas, IVIC. Apartado 20632, Caracas 1020-A, Repu´blica Bolivariana de Venezuela Tel: +58 212 5041526; fax: +58 212 5041086; e-mail: [email protected] Received 27 August 2014 Revised 21 January 2015 Accepted 30 January 2015
(amine donor) residue [7]. Multimeric vWF molecules are not crosslinked to each other by FXIIIa, but can incorporate putrescine, suggesting that only glutamine residues are available for crosslinking [8]. Probably vWF Gln313 and Gln560 residues crosslinked covalently to the fibrin a chains [7,9]. Preferential binding of highmolecular-weight multimers (HMWMs) of vWF occurred with both crosslinked or noncrosslinked fibrin [10]. Preliminary experiments in our laboratory showed that there was a marked difference in the fibrin polymerization process if vWF was crosslinked or not to fibrin. The aim of the present work was to study the effect of vWF on clot structure and fibrin dissolution.
Materials and methods All chemicals were of analytical grade, most of them from Sigma Chemical Company (St. Louis, Missouri, USA). Bovine thrombin was from Sigma Chemical Company. vWF and FXIII were from American Diagnostica (Greenwich, Connecticut, USA). Alexa fluor 488 was purchased from Invitrogen (Nalge Nunc International, Rochester, New York, USA). Factor XIII activation
FXIII was activated essentially as described elsewhere [11]. Briefly, to 72 ml of commercial FXIII (20 mg/ml), 0.1 IU of thrombin, 1.25 U of aprotinin and 228 ml of Tris-buffered saline (TBS) (50 mmol/l Tris, 0.15 NaCl, DOI:10.1097/MBC.0000000000000284
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534 Blood Coagulation and Fibrinolysis 2015, Vol 26 No 5
Fibrin polymerization and fibrinolysis
Fibrinogen was purified by standard protocol using b-alanine as precipitating agent [12], and dialyzed against TBS overnight. The integrity of the purified protein was assessed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE). The contamination of the purified fibrinogen with vWF was quantified by a sandwich ELISA at the Municipal Blood Bank of Caracas (3% and 14% IU/dl were the vWF contamination of the two fibrinogen preparations used in the present work). The fibrin clot was done as follows: to 100 ml of purified fibrinogen (1 mg/ml), 10 ml vWF (5 mg/ml) or TBS (control clot), 5 ml of CaCl2 (5 mmol/l) were added and incubated during 1 min. Then, 20 ml of thrombin–FXIIIa was added (10 ml FXIIIa and 10 ml of 1 IU/ml thrombin). The fibrin formation process was followed by recording the OD each 30 s at 350 nm in an Infinite 200 M (Tecan, Vienna, Austria). After 2.5 h, the polymerization recording was stopped and the plate moved out. Carefully, on the top of each clot was layered 100 ml of plasmin (12 mg/ml). Plate was placed again in the instrument and fibrin dissolution was recorded each 30 s until values returned to baseline. Triplicate of each condition was performed in at least three independent experiments. The absorbance was expressed in mOD (OD 1000). The variables measured were essentially as described elsewhere [13]: lag time (s), slope (mOD/s) and maximum absorbance (MaxAbs; mOD) for fibrin polymerization, and time to 50% clot lysis (Lys50MA in seconds, calculated as the time from MaxAbs to the time at which a 50% reduction of this value occurred) and lysis rate (in mOD/s, calculated as the slope of the curve from MaxAbs to baseline absorbance). Permeation
One hundred microliter purified fibrinogen was mixed with 10 ml of vWF (5 mg/ml) or TBS and 5-ml CaCl2 (5 mmol/l), and incubated for 1 min at room temperature. Then 20 ml of thrombin–FXIIIa (10 ml of 1 IU/ml thrombin and 10 ml of FXIIIa) was added. One hundred microliter of the mixture was immediately transferred inside a plastic column, and the permeation recording was done as described elsewhere [14]. Two independent experiments were performed of four clots each time, weighing 3 drops/ clot. Laser scanning confocal microscopy
Clots were formed with the same clotting conditions as for polymerization. Fibrin was stained with Alexa Fluor 488 conjugated to fibrinogen. The clotting mixture was
prepared in Eppendorf tubes and the reaction was quickly transferred to a glass chamber, allowed to clot for 2 h at 378C. Clot structure was visualized using a LSCM model Nikon C1 (Nikon, Japan), with a laser unit argon-cooled air (488 nm). The objective used was Plan Apo VC 60X of water immersion. For analysis, a Z-stack image was taken from the bottom of the dish (0 mm) up to 30 mm, with step sizes of 0.5 mm and a volume render constructed. Each sample was tested at least in three independent experiments, by duplicate each time. Several fields of each duplicate were examined at random, to obtain fields that were characteristic of the entire sample. Four areas of 212 212 mm (x, y) were selected from each duplicate and digitized. The diameters of fibrin fibers and its density were quantified from the volume render of the images, with the Olympus FV10-ASW 2.1 and Origin Pro 8 software. Statistical analysis
Statistical analysis was performed with the OriginPro program version 8.1. Results are presented as mean ( standard deviation). Kolmorov-Smirnov test was used to study the adjustment of variables to the normal distribution. Means were compared by one-way analysis of variance. A significance level of 0.05 was used.
Results Preliminary data showed that the effect of vWF on fibrin polymerization was more pronounced when no external FXIIIa was added (Fig. 1). VWF had a marked effect on the maximum absorbance (MaxAbs): 612 28 vs. 281 17 mOD, P < 0.05. Furthermore, the time to reach the plateau shortened in the presence of FXIIIa. From Fig. 1
700 600 500
mOD (350 nm)
pH 7.4) supplemented with 5-mmol/l CaCl2 were added. The mixture was incubated at 378C for 1 h. The FXIIIa was aliquoted, and kept at 808C until use.
400 300 200 100 0 0
1000
2000
3000
4000
Time (s) Effect of von Willebrand factor (vWF) on fibrin polymerization. VWF was added to purified fibrinogen and crosslinked to fibrin by activated factor XIII (FXIIIa). Fibrin polymerization without adding external vWF and FXIIIa (D); in the presence of vWF but without FXIIIa &; without vWF but adding external FXIIIa (~); adding both vWF and FXIIIa (&).
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von Willebrand factor and clot structure Marchi and Rojas 535
Fig. 1 it is clearly seen that after 1 h of polymerization clots performed without adding FXIIIa and in the presence of vWF still growing, so the presence of FXIIIa limited the radial growth of the fibrin fibers.
Fig. 2
(a)
It is well established that clot structure modulates the rate of clot dissolution. We found that vWF increased the rate of fibrin formation and final turbidity when it was crosslinked to fibrin (Table 1). External fibrinolysis achieved with plasmin showed that vWF crosslinked to fibrin accelerated four times clot dissolution and shortened the time to dissolve 50% of the clot (Table 1). It seemed that plasmin diffused faster on clots that had incorporated vWF, which could be attributed to a fibrin meshwork with bigger pores and thicker fibers. The permeation experiments confirmed our assumption that the permeation coefficient (Ks) was 2.5 times higher in clots with incorporated vWF (Table 1), and LSCM pictures showed the presence of big pores filling space and thicker fibers on clots that had vWF (1.02 0.01 vs. 0.86 0.02 mm without vWF, P < 0.05) (Fig. 2).
(b)
Discussion Von Willebrand disease (vWD) is the most common inherited bleeding disorder caused by quantitative and/ or qualitative deficiency of vWF. This factor has two major roles in hemostasis, facilitates platelet adhesion to the exposed subendothelium to form a platelet plug and is the carrier protein of procoagulant FVIII, avoiding its premature degradation in the bloodstream [1,15]. vWD has been classified into three major categories: type 1, which is characterized by a partial quantitative deficiency of vWF and accordingly its functional properties; type 2, in which there are qualitative defects affecting vWF function; and type 3, in which vWF is totally deficient. The vWF and FVIII levels determine the bleeding tendency in vWD; however, the bleeding tendency is not completely related to vWF levels. The cause of this large variability in the bleeding tendency in patients with vWD is unknown [16]. Clot stability is related to the fibrin viscoelastic properties and dissolution rate, and can determine a bleeding or Summary of the effect of von Willebrand factor on fibrin polymerization, external fibrinolysis and permeation
Table 1
Polymerization Lag time (s) Slope (mOD/s) MaxAbs (mOD) External fibrinolysis Lys50MA (s) Lysis rate (mOD/s) Permeation Ks (cm2) 10 9
Without vWF
With vWF
0 0.51 0.23 216 51
0 1.47 0.76M 455 149M
22208 6727 0.047 0.018
16732 1566M 0.185 0.082M
8.3 1.1
20.4 1.6M
Results are expressed as the mean (SD). Ks, permeation coefficient or Darcy constant; Lys50MA, interval of time to achieve a 50% reduction of MaxAbs; MaxAbs, maximum absorbance; vWF, von Willebrand factor. M P < 0.05.
Laser scanning confocal microscopy of fibrin clots. Clots formed without adding external von Willbrand factor (vWF) (a) and clots with crosslinked vWF (b).
thrombotic phenotype [5,17]. The viscoelastic fibrin properties and dissolution rate depend on the fibrin structure. Fibrin structure is modulated in vitro and in vivo not only by fibrinogen, thrombin and calcium concentrations or ionic strength, but also by the noncovalent or covalent associations of a great variety of proteins [18,19]. As a result of these conditions (i.e. increasing fibrinogen or thrombin concentration or the binding of proteins, covalent or not), the fibrin network has thicker or thinner fibers, large or small pores and increased or diminished fibrin density that affect the rate of fibrin dissolution. The effect of vWF on clot structure and lysis has not yet been investigated. A recent work describes the effect of fibrinolysis on bleeding phenotype in moderate and severe vWD [20]. The authors measured the clot lysis time (CLT) in a large cohort of vWD patients (type 1, 2 and 3) and correlated it with the bleeding score,
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536 Blood Coagulation and Fibrinolysis 2015, Vol 26 No 5
concluding that the plasma fibrinolytic potential does not influence the bleeding tendency in vWD patients. Unfortunately, a control group of healthy people was not included to establish normal CLT reference values, but interestingly they found prolonged CLTs in type 3 vWD (virtually deficiency of vWF) compared with vWD type 1 and type 2 [20]. These results apparently are in agreement with our work that vWF accelerates fibrin dissolution. The CLTs measured by Wee et al. is the same parameter as Lys50MA (terminology used by us). Although our study is a preliminary work, as our purified fibrinogen was not totally depleted of vWF and FXIII, the effect of vWF on clot structure and fibrin dissolution was significant. It is described in the literature that the HMWMs (5500–10000 kDa) are preferentially bound to crosslinked or noncrosslinked fibrin [10]. The commercial vFW used in the present work had the whole range of vWF multimers (1–20 106 Da), according to the data sheet of the manufacturer. It is quite intuitive that these HMWMs would increase the pore size of fibrin meshwork and fibrin fibers diameter, facilitating the diffusion of fibrinolytic components that resolve the clot. The dissolution strategy used by us that is external fibrinolysis laying plasmin on the top of the clot directly relates clot dissolution with clot structure, as the conversion of plasminogen to plasmin by tissue type plasminogen activator on the fibrin surface is absent. Our study has several limitations. Although the results obtained from polymerization, fibrinolysis, permeation and LSCM were consistent with the effect of FvW on clot structure and fibrinolysis, other tests need to be performed to strengthen these preliminary observations. For example, structural changes on the fibrin network could be visualized at higher resolution by the use of the electron microscope or using different fibrinolytic strategies. Finally, it would be interesting in the future to perform clot structure and dissolution analysis on plasma from vWD patients to evaluate the potential clinical utility.
Acknowledgements We thank BSc. Marion Echenagucia for vWF quantification on purified fibrinogen at the Municipal Blood Bank of Caracas, and to BSc. Marisela De Agrela for her technical assistance.
Conflicts of interest
There are no conflicts of interest.
References 1 2 3 4 5 6
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11
12
13
14
15
16
17 18 19
20
Sadler JE. Biochemistry and genetics of von Willebrand factor. Annu Rev Biochem 1998; 67:395–424. Castaman G. New development in von Willebrand disease. Curr Opin Hematol 2013; 20:424–429. Weisel JW, Litvinov RI. Mechanisms of fibrin polymerization and clinical implications. Blood 2013; 121:1712–1719. Lord ST. Fibrinogen and fibrin: scaffold proteins in hemostasis. Curr Opin Hematol 2007; 14:236–241. Weisel JW. Structure of fibrin: impact on clot stability. J Thromb Haemost 2007; 5 (Suppl 1):116–124. Richardson VR, Schroeder V, Grant PJ, Standeven KF, Carter AM. Complement C3 is a substrate for activated factor XIII that is cross-linked to fibrin during clot formation. Br J Haematol 2013; 160:116–119. Richardson VR, Cordell P, Standeven KF, Carter AM. Substrates of Factor XIII-A: roles in thrombosis and wound healing. Clin Sci (Lond) 2013; 124:123–137. Hada M, Kaminski M, Bockenstedt P, McDonagh J. Covalent crosslinking of von Willebrand factor to fibrin. Blood 1986; 68:95–101. Takagi J, Aoyama T, Ueki S, Ohba H, Saito Y, Lorand L. Identification of factor-XIIIa-reactive glutaminyl residues in the propolypeptide of bovine von Willebrand factor. Eur J Biochem 1995; 232:773–777. Ribes JA, Francis CW. Multimer size dependence of von Willebrand factor binding to crosslinked or noncrosslinked fibrin. Blood 1990; 75:1460–1465. Niwa K, Takebe M, Sugo T, Kawata Y, Mimuro J, Asakura S, et al. A gamma Gly-268 to Glu substitution is responsible for impaired fibrin assembly in a homozygous dysfibrinogen Kurashiki I. Blood 1996; 87:4686–4694. Kierulf P, Jakobsen E. A modified beta-alanine precipitation procedure to prepare fibrinogen free of antithrombin III and plasminogen. Thromb Res 1973; 3:145–148. Marchi R, Garmendia J, Kanzler D, Fuenmayor J. Study of the fibrinolytic process in a patient with antiphospholipid syndrome. Blood Coagul Fibrinolysis 2015; 26:239–245. Pieters M, Undas A, Marchi R, De Maat MP, Weisel J, Arie¨ns RA. An international study on the standardization of fibrin clot permeability measurement: methodological considerations and implications for healthy control values. J Thromb Haemost 2012; 10:2179–2181. Stockschlaeder M, Schneppenheim R, Budde U. Update on von Willebrand factor multimers: focus on high-molecular-weight multimers and their role in hemostasis. Blood Coagul Fibrinolysis 2014; 25:206–216. Tosetto A, Rodeghiero F, Castaman G, Goodeve A, Federici AB, Batlle J, et al. A quantitative analysis of bleeding symptoms in type 1 von Willebrand disease: results from a multicenter European study (MCMDM-1 VWD). J Thromb Haemost 2006; 4:766–773. Lord ST. Molecular mechanisms affecting fibrin structure and stability. Arterioscler Thromb Vasc Biol 2011; 31:494–499. Weisel JW. Fibrinogen and fibrin. Adv Protein Chem 2005; 70:247–299. Marchi R. Fibrinogen: structural and functional aspects. In: Stief T, editor. Thrombin: function and pathophysiolog. New York: Nova Science Publishers; 2012. pp. 263–292. De Wee EM, Klaij K, Eikenboom HC, Van Der Bom JG, Fijnvandraat K, Laros-Van Gorkom BA. Effect of fibrinolysis on bleeding phenotype in moderate and severe von Willebrand disease. Haemophilia 2012; 18:444–451.
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Effect of von Willebrand factor on clot structure and lysis Rita Marchia and He´ctor Rojasb Von Willebrand Factor (vWF) is constitutively secreted by the endothelium and incorporated in the fibrin clots under slow clotting conditions. The aim of the present work was to study the effect of vWF on clot structure and lysis. Purified fibrinogen was mixed with vWF or Tris-buffered saline and clotted with thrombin – activated factor XIII. Fibrin polymerization was followed by turbidity at 350 nm during 2.5 h. After this time, plasmin was added on the top of the clots, and the optical density (OD) was read until baseline values. vWF effect on network´s porosity was evaluated by permeation using the same clotting conditions as for fibrin polymerization. Clot structure was visualized and analyzed by laser scanning confocal microscopy (LSCM). The rate of fibrin polymerization was 1.47 mOD/s in the presence of vWF and 0.5 mOD/s when vWF was not added (P < 0.05). The fibrin lysis rate was approximately four times faster when vWF was added to fibrinogen. The fibrin network porosity was (20.4 W 1.6) T 10S9 cm2 with vWF and (8.3 W 1.2) T 10S9 cm2 without external vWF (P < 0.05). The analysis of LSCM images showed that vWF increased fibrin fibers diameter and the networks´ pores size. In conclusion,
Introduction Von Willebrand factor (vWF) is a large, multimeric plasma glycoprotein that plays an essential role in the initial adhesion of platelets to subendothelial connective tissue at the sites of vascular injury. vWF also is a carrier protein of blood clotting factor VIII (FVIII), protecting it from proteolytic degradation [1], and is assembled from identical subunits of approximately 250 kDa, which range in size from dimers of 500 kDa to multimers more than 20 000 kDa. The functional and/or quantitative vWF abnormalities cause the von Willebrand factor disease (vWFD), which is the most common inherited bleeding disorder [2]. The structure of the clot can be characterized by the fiber diameter, density, number and nature of the branch points (tri or tetramolecular), distances between branch points and size of the pores [3]. A variety of proteins bind to the fibrin clot through noncovalent and/or covalent bonds modifying its structure and the rate of fibrin degradation [4,5]. Actin, fibronectin, vWF and complement C3 [6], and the fibrinolytic inhibitors a2-antiplasmin, plasminogen activator inhibitor 2 (PAI 2) or thrombin activatable fibrinolysis inhibitor (TAFI), are crosslinked to fibrin by activated factor XIII (FXIIIa) [7]. FXIII is a transglutaminase, which catalyzes the formation of covalent e-(g-glutamyl)lysine crosslinks between the g-carboxy-amine group of a glutamine (amine acceptor) and the e-amino group of a lysine 0957-5235 Copyright ß 2015 Wolters Kluwer Health, Inc. All rights reserved.
vWF covalently crosslinked to fibrin modify its structure (increases fibrin diameter and the pores filling space of the meshwork) that accelerates the fibrin lysis rate. Blood Coagul Fibrinolysis 26:533–536 Copyright ß 2015 Wolters Kluwer Health, Inc. All rights reserved.
Blood Coagulation and Fibrinolysis 2015, 26:533–536 Keywords: confocal microscopy, fibrin polymerization, fibrinolysis, permeation, von Willebrand factor a Lab. Biologı´a del Desarrollo de la Hemostasia, IVIC and bInstituto de Inmunologı´a, Universidad Central de Venezuela (UCV), Caracas, Repu´blica Bolivariana de Venezuela
Correspondence to Rita Marchi Cappelletti, Centro de Medicina Experimental, Laboratorio de Fisiopatologı´a, Seccio´n Biologı´a del Desarrollo de la Hemostasia; Instituto Venezolano de Investigaciones, Cientı´ficas, IVIC. Apartado 20632, Caracas 1020-A, Repu´blica Bolivariana de Venezuela Tel: +58 212 5041526; fax: +58 212 5041086; e-mail: [email protected] Received 27 August 2014 Revised 21 January 2015 Accepted 30 January 2015
(amine donor) residue [7]. Multimeric vWF molecules are not crosslinked to each other by FXIIIa, but can incorporate putrescine, suggesting that only glutamine residues are available for crosslinking [8]. Probably vWF Gln313 and Gln560 residues crosslinked covalently to the fibrin a chains [7,9]. Preferential binding of highmolecular-weight multimers (HMWMs) of vWF occurred with both crosslinked or noncrosslinked fibrin [10]. Preliminary experiments in our laboratory showed that there was a marked difference in the fibrin polymerization process if vWF was crosslinked or not to fibrin. The aim of the present work was to study the effect of vWF on clot structure and fibrin dissolution.
Materials and methods All chemicals were of analytical grade, most of them from Sigma Chemical Company (St. Louis, Missouri, USA). Bovine thrombin was from Sigma Chemical Company. vWF and FXIII were from American Diagnostica (Greenwich, Connecticut, USA). Alexa fluor 488 was purchased from Invitrogen (Nalge Nunc International, Rochester, New York, USA). Factor XIII activation
FXIII was activated essentially as described elsewhere [11]. Briefly, to 72 ml of commercial FXIII (20 mg/ml), 0.1 IU of thrombin, 1.25 U of aprotinin and 228 ml of Tris-buffered saline (TBS) (50 mmol/l Tris, 0.15 NaCl, DOI:10.1097/MBC.0000000000000284
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534 Blood Coagulation and Fibrinolysis 2015, Vol 26 No 5
Fibrin polymerization and fibrinolysis
Fibrinogen was purified by standard protocol using b-alanine as precipitating agent [12], and dialyzed against TBS overnight. The integrity of the purified protein was assessed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE). The contamination of the purified fibrinogen with vWF was quantified by a sandwich ELISA at the Municipal Blood Bank of Caracas (3% and 14% IU/dl were the vWF contamination of the two fibrinogen preparations used in the present work). The fibrin clot was done as follows: to 100 ml of purified fibrinogen (1 mg/ml), 10 ml vWF (5 mg/ml) or TBS (control clot), 5 ml of CaCl2 (5 mmol/l) were added and incubated during 1 min. Then, 20 ml of thrombin–FXIIIa was added (10 ml FXIIIa and 10 ml of 1 IU/ml thrombin). The fibrin formation process was followed by recording the OD each 30 s at 350 nm in an Infinite 200 M (Tecan, Vienna, Austria). After 2.5 h, the polymerization recording was stopped and the plate moved out. Carefully, on the top of each clot was layered 100 ml of plasmin (12 mg/ml). Plate was placed again in the instrument and fibrin dissolution was recorded each 30 s until values returned to baseline. Triplicate of each condition was performed in at least three independent experiments. The absorbance was expressed in mOD (OD 1000). The variables measured were essentially as described elsewhere [13]: lag time (s), slope (mOD/s) and maximum absorbance (MaxAbs; mOD) for fibrin polymerization, and time to 50% clot lysis (Lys50MA in seconds, calculated as the time from MaxAbs to the time at which a 50% reduction of this value occurred) and lysis rate (in mOD/s, calculated as the slope of the curve from MaxAbs to baseline absorbance). Permeation
One hundred microliter purified fibrinogen was mixed with 10 ml of vWF (5 mg/ml) or TBS and 5-ml CaCl2 (5 mmol/l), and incubated for 1 min at room temperature. Then 20 ml of thrombin–FXIIIa (10 ml of 1 IU/ml thrombin and 10 ml of FXIIIa) was added. One hundred microliter of the mixture was immediately transferred inside a plastic column, and the permeation recording was done as described elsewhere [14]. Two independent experiments were performed of four clots each time, weighing 3 drops/ clot. Laser scanning confocal microscopy
Clots were formed with the same clotting conditions as for polymerization. Fibrin was stained with Alexa Fluor 488 conjugated to fibrinogen. The clotting mixture was
prepared in Eppendorf tubes and the reaction was quickly transferred to a glass chamber, allowed to clot for 2 h at 378C. Clot structure was visualized using a LSCM model Nikon C1 (Nikon, Japan), with a laser unit argon-cooled air (488 nm). The objective used was Plan Apo VC 60X of water immersion. For analysis, a Z-stack image was taken from the bottom of the dish (0 mm) up to 30 mm, with step sizes of 0.5 mm and a volume render constructed. Each sample was tested at least in three independent experiments, by duplicate each time. Several fields of each duplicate were examined at random, to obtain fields that were characteristic of the entire sample. Four areas of 212 212 mm (x, y) were selected from each duplicate and digitized. The diameters of fibrin fibers and its density were quantified from the volume render of the images, with the Olympus FV10-ASW 2.1 and Origin Pro 8 software. Statistical analysis
Statistical analysis was performed with the OriginPro program version 8.1. Results are presented as mean ( standard deviation). Kolmorov-Smirnov test was used to study the adjustment of variables to the normal distribution. Means were compared by one-way analysis of variance. A significance level of 0.05 was used.
Results Preliminary data showed that the effect of vWF on fibrin polymerization was more pronounced when no external FXIIIa was added (Fig. 1). VWF had a marked effect on the maximum absorbance (MaxAbs): 612 28 vs. 281 17 mOD, P < 0.05. Furthermore, the time to reach the plateau shortened in the presence of FXIIIa. From Fig. 1
700 600 500
mOD (350 nm)
pH 7.4) supplemented with 5-mmol/l CaCl2 were added. The mixture was incubated at 378C for 1 h. The FXIIIa was aliquoted, and kept at 808C until use.
400 300 200 100 0 0
1000
2000
3000
4000
Time (s) Effect of von Willebrand factor (vWF) on fibrin polymerization. VWF was added to purified fibrinogen and crosslinked to fibrin by activated factor XIII (FXIIIa). Fibrin polymerization without adding external vWF and FXIIIa (D); in the presence of vWF but without FXIIIa &; without vWF but adding external FXIIIa (~); adding both vWF and FXIIIa (&).
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von Willebrand factor and clot structure Marchi and Rojas 535
Fig. 1 it is clearly seen that after 1 h of polymerization clots performed without adding FXIIIa and in the presence of vWF still growing, so the presence of FXIIIa limited the radial growth of the fibrin fibers.
Fig. 2
(a)
It is well established that clot structure modulates the rate of clot dissolution. We found that vWF increased the rate of fibrin formation and final turbidity when it was crosslinked to fibrin (Table 1). External fibrinolysis achieved with plasmin showed that vWF crosslinked to fibrin accelerated four times clot dissolution and shortened the time to dissolve 50% of the clot (Table 1). It seemed that plasmin diffused faster on clots that had incorporated vWF, which could be attributed to a fibrin meshwork with bigger pores and thicker fibers. The permeation experiments confirmed our assumption that the permeation coefficient (Ks) was 2.5 times higher in clots with incorporated vWF (Table 1), and LSCM pictures showed the presence of big pores filling space and thicker fibers on clots that had vWF (1.02 0.01 vs. 0.86 0.02 mm without vWF, P < 0.05) (Fig. 2).
(b)
Discussion Von Willebrand disease (vWD) is the most common inherited bleeding disorder caused by quantitative and/ or qualitative deficiency of vWF. This factor has two major roles in hemostasis, facilitates platelet adhesion to the exposed subendothelium to form a platelet plug and is the carrier protein of procoagulant FVIII, avoiding its premature degradation in the bloodstream [1,15]. vWD has been classified into three major categories: type 1, which is characterized by a partial quantitative deficiency of vWF and accordingly its functional properties; type 2, in which there are qualitative defects affecting vWF function; and type 3, in which vWF is totally deficient. The vWF and FVIII levels determine the bleeding tendency in vWD; however, the bleeding tendency is not completely related to vWF levels. The cause of this large variability in the bleeding tendency in patients with vWD is unknown [16]. Clot stability is related to the fibrin viscoelastic properties and dissolution rate, and can determine a bleeding or Summary of the effect of von Willebrand factor on fibrin polymerization, external fibrinolysis and permeation
Table 1
Polymerization Lag time (s) Slope (mOD/s) MaxAbs (mOD) External fibrinolysis Lys50MA (s) Lysis rate (mOD/s) Permeation Ks (cm2) 10 9
Without vWF
With vWF
0 0.51 0.23 216 51
0 1.47 0.76M 455 149M
22208 6727 0.047 0.018
16732 1566M 0.185 0.082M
8.3 1.1
20.4 1.6M
Results are expressed as the mean (SD). Ks, permeation coefficient or Darcy constant; Lys50MA, interval of time to achieve a 50% reduction of MaxAbs; MaxAbs, maximum absorbance; vWF, von Willebrand factor. M P < 0.05.
Laser scanning confocal microscopy of fibrin clots. Clots formed without adding external von Willbrand factor (vWF) (a) and clots with crosslinked vWF (b).
thrombotic phenotype [5,17]. The viscoelastic fibrin properties and dissolution rate depend on the fibrin structure. Fibrin structure is modulated in vitro and in vivo not only by fibrinogen, thrombin and calcium concentrations or ionic strength, but also by the noncovalent or covalent associations of a great variety of proteins [18,19]. As a result of these conditions (i.e. increasing fibrinogen or thrombin concentration or the binding of proteins, covalent or not), the fibrin network has thicker or thinner fibers, large or small pores and increased or diminished fibrin density that affect the rate of fibrin dissolution. The effect of vWF on clot structure and lysis has not yet been investigated. A recent work describes the effect of fibrinolysis on bleeding phenotype in moderate and severe vWD [20]. The authors measured the clot lysis time (CLT) in a large cohort of vWD patients (type 1, 2 and 3) and correlated it with the bleeding score,
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536 Blood Coagulation and Fibrinolysis 2015, Vol 26 No 5
concluding that the plasma fibrinolytic potential does not influence the bleeding tendency in vWD patients. Unfortunately, a control group of healthy people was not included to establish normal CLT reference values, but interestingly they found prolonged CLTs in type 3 vWD (virtually deficiency of vWF) compared with vWD type 1 and type 2 [20]. These results apparently are in agreement with our work that vWF accelerates fibrin dissolution. The CLTs measured by Wee et al. is the same parameter as Lys50MA (terminology used by us). Although our study is a preliminary work, as our purified fibrinogen was not totally depleted of vWF and FXIII, the effect of vWF on clot structure and fibrin dissolution was significant. It is described in the literature that the HMWMs (5500–10000 kDa) are preferentially bound to crosslinked or noncrosslinked fibrin [10]. The commercial vFW used in the present work had the whole range of vWF multimers (1–20 106 Da), according to the data sheet of the manufacturer. It is quite intuitive that these HMWMs would increase the pore size of fibrin meshwork and fibrin fibers diameter, facilitating the diffusion of fibrinolytic components that resolve the clot. The dissolution strategy used by us that is external fibrinolysis laying plasmin on the top of the clot directly relates clot dissolution with clot structure, as the conversion of plasminogen to plasmin by tissue type plasminogen activator on the fibrin surface is absent. Our study has several limitations. Although the results obtained from polymerization, fibrinolysis, permeation and LSCM were consistent with the effect of FvW on clot structure and fibrinolysis, other tests need to be performed to strengthen these preliminary observations. For example, structural changes on the fibrin network could be visualized at higher resolution by the use of the electron microscope or using different fibrinolytic strategies. Finally, it would be interesting in the future to perform clot structure and dissolution analysis on plasma from vWD patients to evaluate the potential clinical utility.
Acknowledgements We thank BSc. Marion Echenagucia for vWF quantification on purified fibrinogen at the Municipal Blood Bank of Caracas, and to BSc. Marisela De Agrela for her technical assistance.
Conflicts of interest
There are no conflicts of interest.
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