Bleeding disorders and thrombotic complications constitute a major cause of death and disability worldwide. Although it is known that the complement and coagulation systems interact, no studies have investigated the specific role or mechanisms of lectin-mediated coagulation in vivo. FeCl3 treatment resulted in intra-arterial occlusive thrombogenesis within 10 min in wild-type (WT) and C2/factor B-null mice. In contrast, mannose-binding lectin (MBL)-null and MBL-associated serine protease (MASP)-1/-3 knockout (KO) mice had significantly decreased FeCl3-induced thrombogenesis. Reconstitution with recombinant human (rh) MBL restored FeCl3-induced thrombogenesis in MBL-null mice to levels comparable to WT mice, suggesting a significant role of the MBL/MASP complex for in vivo coagulation. Additionally, whole blood aggregation demonstrated increased MBL/MASP complex-dependent platelet aggregation. In vitro, MBL/MASP complexes were captured on mannan-coated plates, and cleavage of a chromogenic thrombin substrate (S2238) was measured. We observed no significant differences in S2238 cleavage between WT, C2/factor B-null, MBL-A−/−, or MBL-C−/− sera; however, MBL-null or MASP-1/-3 KO mouse sera demonstrated significantly decreased S2238 cleavage. rhMBL alone failed to cleave S2238, but cleavage was restored when rMASP-1 was added to either MASP-1/-3 KO sera or rhMBL. Taken together, these findings indicate that MBL/MASP complexes, and specifically MASP-1, play a key role in thrombus formation in vitro and in vivo.
Occlusive thrombosis, resulting from atherosclerotic plaque rupture or restenosis, plays an important role in the onset of two major causes of death in developed nations: myocardial infarction and ischemic stroke. Typically, acute thrombolytic therapy is the first step taken to reperfuse ischemic tissues downstream of the thrombus. Additionally, most patients suffering from cardiovascular disease are prescribed chronic prophylactic thrombolytic drugs. Coagulation is a complex process and there is a delicate balance between bleeding disorders and clot formation. Considering the widespread and critical role of thrombosis in many diseases, significant research efforts have been aimed at the discovery and development of safe and effective antithrombotic (e.g., anti-platelet, anti-thrombin) drugs for acute and chronic clinical use.
The complement cascade is part of the innate immune system responsible for the initiation of inflammation and elimination of invading foreign cells. There are three independent pathways that can initiate the complement cascade: the classical, alternative, and lectin pathways. All three pathways converge at formation of the C3 convertase and follow a common pathway resulting in the formation of C5b-9 (the membrane attack complex). Lectin pathway activation is initiated via the binding of a multimolecule complex to carbohydrate structures present on pathogens, or glycosylation patterns on apoptotic, necrotic, or ischemic cells (1–3). The mannose-binding lectin (MBL) complex consists of MBL and three serine proteases, MBL-associated serine proteases (MASPs) 1, 2, and 3, as well as MAp19 and Map44/MAP-1, two nonenzymatic, truncated products of the MASP2 gene, which may regulate lectin pathway activation by competing for the binding of MASPs to the carbohydrate recognition complexes (2–10). The system is slightly altered in mice, where two forms of MBL exist, MBL-A and MBL-C (2, 7).
Coagulation, like complement, is a highly conserved cascade-style system composed of multiple factors that are activated in a sequential and amplified process, ultimately resulting in the formation of an insoluble fibrin clot. The coagulation cascade is primarily responsible for maintaining hemostasis following vascular injury. Activation of either the intrinsic or extrinsic pathways that make up the coagulation cascade leads to the formation of a prothrombinase complex composed of factors Va and Xa, cleaving prothrombin to thrombin. Thrombin formation is a critical central step in coagulation, cleaving fibrinogen, factor XIII, and activating platelets. Thrombin also plays important roles in the activation of protein C, an anti-coagulative protein with cellular protective actions (11, 12).
For many years, it has been recognized that the complement and coagulation systems interact (13). Complement activation is known to contribute to thrombotic tissue injury in systemic lupus erythematosus (14), biomaterial-associated thrombosis (15), and paroxysmal nocturnal hemoglobinuria (16), to name just a few. Additionally, reversal of heparinization with protamine, TPA, and streptokinase activate complement (17–19). An important study by Huber-Lang et al. (20) demonstrated that thrombin can directly activate C5 to produce C5a and C5b-9 in C3-deficient mice. Furthermore, MASP-2 can activate prothrombin to thrombin and may explain the mechanism by which thrombin is produced from prothrombin in C3-deficient mice (21). More recently, work from our group indicated that MBL/MASP complexes are associated with thrombin-like activity in vitro and found that MBL-null mice have prolonged bleeding times in vivo (22).
Endothelial injury and platelet aggregation are key events in the pathogenesis of thrombus formation. Our experimental approach was based on the hypothesis that occlusive thrombosis can be initiated by FeCl3-induced endothelial injury, likely mediated by oxidative radical formation and/or transported intraluminal ferric ion interaction with platelets, fibrin, and other formed blood elements (23). For in vivo analysis, the FeCl3 method is the optimal technique, as it exhibits many features useful for studying the molecular determinants of arterial thrombosis in transgenic mice, including uniform injury from animal to animal, and the duration of injury can be precisely controlled. Additionally, whole blood aggregation has been extensively used to elucidate and characterize various signaling pathways and pharmaceuticals involved in platelet stimulation and activation (24, 25). Thus, our repertoire of complement-deficient mice (all on a CH57BL/6 background), along with clinically relevant coagulation models, allows us to effectively address the role of complement in coagulation.
In this study, we report that MBL complexes, and specifically MASP-1, have thrombin-like activity and are a significant regulator of thrombus formation both in vitro and in vivo. The results demonstrate that the innate immune system (complement in particular) has important interactions with the coagulation system previously not appreciated. It is proposed that this mechanism may represent a novel nonimmunological role for the MBL/MASP complex in hemostasis and coagulopathy.
Materials and Methods
All procedures were reviewed and approved by the Institute for Laboratory Animal Research Guide for Care and Use of Laboratory Animals. Wild-type (WT), MBL-A/-C–null, C2/factor B (C2/fB)-null (26), and MASP-1/-3 knockout (KO) (27) mice raised on a C57BL/6 background were used for the present study. Previous reports found that clotting parameters (prothrombin time, activated partial thromboplastin time, fibrinogen, and platelets) in MBL-null mice are similar to WT mice, indicating that coagulation itself is not compromised in MBL deficiency (22).
FeCl3 coagulation model
We used a mouse model of localized thrombus formation as previously described (22, 23, 28). After isoflurane anesthesia, mice were shaved and prepared for FeCl3-induced arterial thrombosis (23). Briefly, an incision was made superficial to the right common carotid artery and an arterial segment was exposed by blunt dissection. Carotid blood flow was measured with a miniature Doppler flow probe (Transonic) as described (29). Localized thrombosis was initiated by applying two pieces of Whatman filter paper saturated with 3.5% FeCl3 to either side of the carotid artery, proximal to the Doppler flow probe. The filter paper was removed 3 min after application, and carotid blood flow was measured continuously for 30 min. The carotid arteries (right and left) were collected at euthanasia for further analysis by immunohistochemistry.
Lectin complement pathway restoration in vivo
Mice were exsanguinated via cardiac puncture and the whole blood samples were allowed to clot for 2 h at room temperature then placed at 4°C overnight. The following day, the clotted samples were centrifuged at 3000 × g for 10 min and the serum was collected, aliquoted, and stored at −80°C for future use.
MBL-dependent whole blood aggregation
Aggregation was analyzed using a whole blood aggregometer (Chrono-log, Havertown, PA) as described previously with slight modifications (24). Mannan (5 mg/ml in NaHCO3/Na2CO3, pH 9.6) was bound to cuvettes (Chrono-log) overnight at 4°C as described (32). The following day, cuvettes were blocked with 3% BSA for 2 h at room temperature then washed with PBS plus 0.05% Tween, PBS, and Veronal-buffered saline. Negative control cuvettes were made by omitting the mannan from the initial plating step. Blood from individual animals was tested in both control and mannan-coated cuvettes and run simultaneously to ensure equal platelet counts. Aggregometry was performed at 37°C with constant stirring. Blood samples were collected in sodium citrate from anesthetized mice via the inferior vena cava. Whole blood was then diluted (1:4) with 0.9% saline and placed in either control or mannan-coated cuvettes. The blood was incubated for 5 min at 37°C, and the aggregation probe was then immersed in the diluted blood sample and incubated at 37°C for 2 min. Aggregation was induced via 1 μg/ml collagen (Chrono-log). Aggregation (ohms of electrical impedance) was recorded for 25 min and all experiments were performed within 3 h of blood acquisition. Data are expressed as the difference between the impedance measured in the mannan-coated cuvette and that in the control cuvette.
Thrombin substrate cleavage assay
The MBL/MASP complex capture assay has been described previously (1) and recently enhanced to a multiplexed system (32). A variation of this assay was developed for the mouse system. Briefly, we captured mouse MBL/MASP complexes on mannan-coated plates for in vitro analysis of complement and coagulation interactions. To examine the individual components of the MBL/MASP complex in the absence of other serum components, we captured rhMBL and rMASP-1 (gift from Dr. Minoru Takahashi) (27). Plates were washed to remove sera components and retain MBL complexes, and a synthetic chromogenic thrombin substrate (S2238; 25 mM; DiaPharma) was added to each well. As S2238 is enzymatically cleaved, p-nitroaniline is released. Thus, the rate of p-nitroaniline formation is proportional to the enzymatic activity. A SpectraMax Plus spectrophotometer (Molecular Diagnostics) was used to measure OD every 5 min for 1.5 h at 405 nm. Data are expressed as the normalized OD.
Following experimental thrombosis, one set of carotid artery sections was removed and placed in formalin. Samples were paraffin embedded and sectioned by AML Laboratories (Baltimore, MD). Thrombus formation was examined via H&E staining as described (33). Additional carotid artery sections from a second group of mice were removed and quickly embedded in OCT and frozen in 2-methylbutane chilled in liquid nitrogen. Frozen sections (5 μm) were stained for MBL using monoclonal rat anti-mouse MBL-A and MBL-C Abs (Hycult Biotech, Uden, The Netherlands) (1:100 in PBS/0.05% Triton X-100 supplemented with 1 mM CaCl2) or mouse monoclonal anti-human 2A9 (anti-hMBL; 1:2000). Briefly, slides were air dried after sectioning. Tissues were fixed with 4% paraformaldehyde for 10 min and rinsed with PBS, followed by incubation with monoclonal rat anti-mouse MBL-A and MBL-C for 1 h. Slides were rinsed with PBS/0.05% Triton X-100, and MBL Ab was detected by biotinylated polyclonal rabbit anti-rat IgG or goat anti-mouse IgG (Dako, Carpinteria, CA) (1:600 in PBS for 45 min). All slides were incubated with a Vectastain ABC-AP kit (standard) (Vector Laboratories, Burlingame, CA), and MBL deposition was visualized using a Vector Red alkaline phosphatase ubstrate Kit (Vector Laboratories). Images were taken on a Nikon Eclipse E400 microscope and analyzed using SPOT imaging software (Diagnostic Instruments). Negative control staining was performed as described above with omission of the primary Ab.
All values are expressed as means ± SEM. Comparisons between groups were made using RM-ANOVA (in vivo studies) or t test (in vitro studies) followed by post hoc analysis. Differences were considered significant at p ≤ 0.05. SigmaStat 3.1 software (Jandel Scientific) was used for statistical analysis.
FeCl3 exposure results in reproducible thrombus formation within the carotid artery (23, 28). As shown in Fig. 1, 3.5% FeCl3 application resulted in complete loss of carotid artery blood flow after ∼10 min in WT mice. Similarly, C2/fB-null mice responded with complete loss of blood flow by 20 min, suggesting that downstream complement activation (C3; C5b-9) plays no role in thrombus formation. In contrast, we observed no decrease in blood flow in MBL-null or MASP-1/-3 mice following FeCl3 exposure (Fig. 1A; p < 0.01). Furthermore, reconstitution with rhMBL (75 μg) in MBL-null mice restored thrombogenesis, supporting a significant role for the MBL/MASP complex in coagulation in vivo.
Carotid arteries were fixed and sectioned transluminally to visualize the thrombus composition. H&E staining shows a distinct thrombus in sections from WT mice following FeCl3 exposure, whereas very little thrombus formation is observed in arteries from MBL-null mice (Fig. 1B). When the lectin pathway was restored with rhMBL in MBL-null mice, thrombus formation was again observed. Similar to MBL-null arteries, MASP-1/-3 KO arteries demonstrated no thrombi, suggesting that a complete MBL/MASP complex is necessary for thrombogenesis following FeCl3 exposure. These results strongly coincide with the in vivo carotid blood flow measurements.
MBL deposition on the vascular endothelium
To determine whether MBL deposition occurred in the regions of FeCl3-initiated thrombus formation, we stained arterial sections for MBL-A and MBL-C. Fig. 2 illustrates MBL deposition along the vascular endothelium in WT and C2/fB-null arteries. As anticipated, no MBL staining is present on the endothelium of MBL-null arteries, but reconstitution with rhMBL again results in MBL deposition and thrombus formation. This strongly supports the FeCl3 findings in C2/fB-null mice, in that the C2/fB-null mice cannot activate downstream complement components (C3; C5b-9) yet we still observed occlusive thrombogenesis. No MBL staining was observed in contralateral arteries or arteries treated with vehicle (data not shown). Thus, our data indicate that the complement components necessary for thrombogenesis are within the MBL/MASP complex alone, and downstream complement activation has little to no role in the thrombogenesis.
MBL-dependent whole blood aggregation
To determine whether lectin complement pathway activation results in increased platelet aggregation, we tested blood samples from WT and MBL-null mice using a Chrono-log whole blood aggregometer in the presence or absence of immobilized mannan. The increase in impedance correlates with the amount of platelet aggregates deposited on the electrodes after the addition of a platelet agonist, in our case, collagen. Fig. 3 demonstrates significantly increased platelet aggregation in whole blood samples from WT mice placed in mannan-coated cuvettes compared with control cuvettes, suggesting that MBL activation on the internal surface of the cuvette enhances the thrombotic potential of the blood sample, therefore leading to increased platelet aggregation. In contrast, there is no significant difference in the maximum aggregation in MBL-null mouse blood incubated in control or mannan-coated cuvettes, indicating that the enhanced aggregation observed in WT blood in mannan-coated cuvettes is mediated by the MBL/MASP complex.
In vitro thrombin-substrate cleavage assays
Following our in vivo observations, we used sera from mice deficient in specific complement components to elucidate the molecular mechanisms responsible for MBL/MASP complex-mediated coagulopathy. Fig. 4 demonstrates that capture of MBL/MASP complexes from MBL-null mouse serum cleaves significantly less thrombin substrate (S2238) compared with WT serum, likely resulting from no MBL available for capture. Capture of MBL-A or MBL-C complexes from respective MBL-C−/− and MBL-A−/− mouse sera results in no significant difference in S2238 cleavage compared with WT mouse sera (Fig. 4), as these genotypes are capable of forming a complete MBL/MASP complex. Furthermore, serum (and thus MBL/MASP complexes) from C2/fB-null mice resulted in S2238 cleavage similar to serum from WT mice, indicating that downstream complement components (i.e., C3, C5b-9) do not play a role in thrombin substrate cleavage. N-acetylglucosamine (GlcNAc) is a competitive inhibitor of MBL binding to mannan, thereby serving as a negative control. When serum from WT mice is treated with GlcNAc (100 mM), MBL complexes do not bind to the microtiter plate and S2238 cleavage is significantly inhibited. These data suggest that an intact MBL/MASP complex is needed for thrombin-like activity in the mouse in vitro system.
To tease out whether the thrombin-like activity was mediated by either MBL or MASPs, we tested S2238 cleavage using MASP-1/-3 KO serum and rMASP-1. No thrombin-like activity was observed in MBL/MASP complexes captured from MASP-1/-3 KO serum (Fig. 5A; p < 0.05) compared with WT sera. However, when rMASP-1 was added to MBL complexes captured from MASP-1/-3 KO serum, thrombin-like activity was restored in a dose-related manner. Furthermore, we observed no thrombin substrate cleavage from the capture of rhMBL alone, but when rMASP1 was coincubated with rhMBL, thrombin-like activity was again observed (Fig. 5B). These data indicate that within the MBL/MASP complex, MASP-1 is the necessary complement component for thrombin-like activity in vitro.
MBL is an interesting complex whereby deficiency in humans is associated with severe atherosclerosis and recurrent infections (34, 35). Our earlier studies demonstrated that ischemic injury results in complement activation and MBL deposition within the vasculature (36, 37). Ischemic injury in a variety of vascular beds initiates lectin-complement pathway activation, and inhibition of MBL or its removal reduces postischemic myocardial reperfusion injury (31, 38). Thus, MBL appears to be a double-edged sword in human disease, where the disease dictates whether the presence or absence of MBL influences the clinical outcome. The present study advances our previous findings to suggest that MBL/MASP complexes play a significant role in thrombus formation in vivo.
MASP-2 can cleave prothrombin to thrombin in vitro (21). Other studies report that MASP-1 may also have thrombin-like activity, as it was found to cleave fibrinogen and factor XIII and lead to a fibrin clot in vitro (39–42). More recently, Takahashi et al. (43) demonstrated that both MASP-1 and thrombin cleaved a synthetic substrate identical to the activation peptide of factor D, further linking MASP-1 to thrombin-like activity. Furthermore, Megyeri et al. (44) demonstrated that MASP-1 activates PAR4, an interesting finding since PAR4 activation by thrombin is responsible for platelet activation in humans (45). However, all of these previous studies were conducted in vitro, and the present studies were undertaken to observe whether these procoagulant activities occur in vivo. These findings may represent a mechanism by which FeCl3-induced thrombogenesis is attenuated in the MBL-null and MASP-1/-3 KO mice, as shown in Fig. 1. Furthermore, these findings demonstrate that a MBL complex comprised of only MASP-2, which is present in the MBL complex of MASP-1/3 KO mice, does not play a sufficient role to induced occlusive thrombosis in vivo in this model.
Westrick et al. (28) published a thorough review of various murine models for examining thrombosis and hemostasis, a significant number of which use the FeCl3 method. FeCl3 was first discovered to be a thrombogenic agent by Reimann-Hunziker in 1944 (46) and has since been widely used to investigate specific mechanisms involved in thrombosis. Many studies suggest that the strongly oxidizing FeCl3 compound injures the endothelial cell wall via oxidative stress (47). Tseng et al. (48) demonstrated that ferric ions permeate the endothelial basal lamina before entering the arterial lumen via an endocytic/exocytic pathway, ultimately resulting in complete endothelial denudation and occlusive thrombosis. Furthermore, Saldeen et al. (49) found that antioxidant therapy significantly decreased FeCl3-induced thrombogenesis. Along these lines, we have previously shown that intracellular oxidative stress leads to MBL ligand expression, MBL complex binding, and complement activation in vitro (1). Thus, the oxidative stress produced by FeCl3 administration likely results in MBL ligand expression and MBL deposition along the vascular endothelium as we have shown (Fig. 2).
We demonstrate that downstream complement activation is not necessary for thrombin-like activity in vitro or in vivo, as demonstrated by the similar results observed between the WT and C2/fB-null mice since the C2/fB-null mice cannot activate complement components C3 through the C5b-9 membrane attack complex. Furthermore, we demonstrate occlusive thrombogenesis in rhMBL reconstituted MBL-null mice in vivo (Fig. 1). These data suggest that MBL inhibition or deficiency decreases coagulation parameters, whereas other complement inhibitors that act on components below MBL in the complement cascade would not attenuate or eliminate coagulation induced by MBL. This hypothesis might explain why a randomized clinical trial involving pexelizumab, a recombinant anti-C5 mAb, in coronary artery bypass graft surgery did not have a statistically significant effect on the primary endpoint (composite endpoint of death or myocardial infarction) (50). Although MBL levels were not measured during these clinical trials, low MBL levels were correlated to a significant reduction in mortality in the Assessment of Pexelizumab in Acute Myocardial Infarction trial (51), suggesting an important role of MBL following myocardial ischemia/reperfusion in this clinical trial. Furthermore, these data suggest that inhibition of the MBL complex and/or MASP-1 will prevent MBL pathway-mediated coagulation, whereas inhibition of MASP-2 would be ineffective (52), as Takahashi et al. (27) reported that MASP-1 is required for enhanced MASP-2–dependent complement activation through direct cleavage and activation of MASP-2.
MBL binding activates the lectin complement pathway, resulting in a pathophysiologic state that may be worse in high-MBL–expressing patients/mice compared with MBL-deficient patients/mice. Furthermore, coagulopathy may result from direct MBL complex-mediated activation of thrombin substrates and coagulation factors. The in vitro and in vivo data presented in this study support this hypothesis. In particular, by capturing and activating MBL complexes on the internal surface of mannan-coated cuvettes used for whole blood aggregation, we demonstrated a significant increase in platelet aggregation compared with control cuvettes in WT mouse blood. No difference in platelet aggregation was observed between the negative control cuvette and the mannan-coated cuvette using MBL-null mouse blood, as would be expected in the absence of MBL complexes. These data further support the in vitro thrombin substrate data suggesting that MASP-1 may be the active component of the MBL complex responsible for thrombin-like activity.
Based on published in vitro observations and the findings obtained in the current study, we propose the following model for FeCl3-mediated occlusive thrombogenesis (Fig. 6). External FeCl3 application results in the translocation of ferric ions and reactive oxygen species via Fenton chemistry, whereby the ferrous iron is oxidized by hydrogen peroxide to form ferric iron, a hydroxyl radical and a hydroxyl anion (47, 48). This hypothesis is further supported by limited thrombogenesis in this model following antioxidant therapy (49). We hypothesize that during the immediate acute phases of endothelial injury, such as FeCl3 application or ischemia/reperfusion, MBL binds to stressed endothelial cells. Indeed, our laboratory demonstrated that stressed endothelial cells activate the lectin complement pathway via ROS generation and induction of an MBL ligand (1, 53, 54). Upon MBL binding to injured endothelial cells, we hypothesize the serine proteases MASP-1 and MASP-2 play a significant role in the amplification of coagulation via the cleavage of prothrombin to thrombin (21), the direct cleavage of thrombin substrates including fibrinogen and factor XIII (41–43), and activation of platelets through PAR4 (44, 45). This sequence of events ultimately results in thrombus formation in vivo. Furthermore, to our knowledge, the data presented in this study represent the first evidence that MBL and/or MASP-1 deficiency leads to coagulation abnormalities in vivo.
Results from this study may have important implications for the early identification, treatment, and prevention of common vascular complications such as myocardial infarction and stroke. Our data extend previously published in vitro findings and show further interactions of lectins (MBL complexes) to activate components of the coagulation system both in vitro and in vivo. The data yield new information on the importance of the MBL complex alone in mediating pathologies without the need for complement activation per se.
We acknowledge the expert technical assistance of Margaret Morrissey during the course of the studies.
This work was supported in part by National Institutes of Health Grants HL056086, HL099130, and AI089781. L.R.L.B. is funded by Ruth L. Kirschstein National Research Service Award Fellowship HL099043.
Abbreviations used in this article:
mannose-binding lectin-associated serine protease
The authors have no financial conflicts of interest.