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Am J Physiol Heart Circ Physiol 292: H2959-H2965, 2007. First published February 9, 2007; doi:10.1152/ajpheart.00730.2006
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Antithrombotic activity of kininogen is mediated by inhibitory effects of domain 3 during arterial injury in vivo

Sarmina Hassan,1 Irma M. Sainz,1 Mohammad M. Khan,1 Harlan N. Bradford,1 Irma Isordia-Salas,1 Sakeen W. Kashem,1 R. Balfour Sartor,2 and Robert W. Colman1

1Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania; and 2Center for Gastrointestinal Biology and Disease, University of North Carolina School of Medicine, Chapel Hill, North Carolina

Submitted 7 July 2006 ; accepted in final form 7 February 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
High-molecular-weight kininogen (HK) and its domain 3 (D3) exhibit anticoagulant properties and inhibit platelet activation at low thrombin concentration in vitro. We hypothesized that the rapid occlusive thrombosis in HK-deficient (HKd) rats following endothelial injury of the aorta results from enhanced platelet aggregation by thrombin. The effects of D3 (G235-M357) or D3-derived peptides on thrombosis in vivo were tested. D3 and its exon 7C terminal peptide (E7CP, K270-Q292), expressed as glutathione S-transferase (GST) fusion proteins (GST-D3, GST-E7CP), or GST alone, as well as cleaved HK (HKa) or synthetic peptide E7CP, were infused intravenously 10 min before endothelial injury. Blood flow was reduced down to 10% of baseline flow within 28 ± 5.2 min by a platelet-fibrin thrombus in GST-treated HKd rats compared with >240 min in GST-treated normal HK rats (wild type). GST-D3, GST-E7CP, HKa, or E7CP infusion prolonged the flow time to 233, >240, 223, and >240 min, respectively, in HKd rats. When GST-E7CP was infused 10 min after the injury, blood flow was maintained for >240 min. Thrombin-antithrombin concentrations were elevated by injury in HKd rats receiving GST from 35 to 55 µg/l and decreased with GST-E7CP, HKa, or E7CP reconstitution to 40, 15, and 9 µg/l, respectively. We conclude that HKd rats are prothrombotic and that HKa, kininogen D3, and its fragment E7CP modulate arterial thrombosis after endothelial injury.

arterial thrombosis; endothelial injury; kininogen-deficient rat

HIGH-MOLECULAR-WEIGHT KININOGEN (HK) and low-molecular-weight kininogen (LK) are multifunctional plasma proteins that are substrates for plasma kallikrein and tissue kallikrein yielding bradykinin and kallidin, respectively. The remaining cleaved HK (HKa), lacking bradykinin, exhibits antiadhesive (26) and antiangiogenic (5) activity as well as enhancing cell-associated fibrinolysis (6) and releasing cytokines and chemokines to enhance inflammation (15). Although deficiency of HK results in vitro in a prolonged activated partial thromboplastin time, patients with plasma HK deficiency do not have a hemorrhagic diathesis (4). Whether individuals with total kininogen deficiency are thrombophilic has not been determined because of the rarity of the disorder. However, both kininogens bind to platelets (9, 19, 25) and selectively inhibit the high-affinity thrombin binding to platelet glycoprotein (GP)Ib (14, 16). Therefore, animal models are the only modality currently available to resolve this issue.

Kininogens are proteins composed of multiple domains (Fig. 1A), each with associated functional activities. The heavy chain, domain (D)1–D3, and bradykinin contained in D4 are common to both HK and LK, while the light chains of each are unique (25). D3 contains amino acid sequences that compete with thrombin for binding to platelet GPIb (1).


Figure 1
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  		Fig. 1. Schematic diagram of kininogens, domains, and fusion proteins. A: high-molecular-weight kininogen (HK) comprised of domain (D)1–D6. D4 of HK contains bradykinin, which is released on cleavage of HK to cleaved HK (HKa) (by kallikrein). Fusion proteins glutathione S-transferase (GST)-D3 and GST-exon 7c terminal peptide (E7CP) are shown. B: D3 exon 7, 8, and 9 products are shown. Shaded area is the E7CP polypeptide.

 
In mice, HK is coded by two functional genes (2); thus animals with selective targeted disruption of the HK gene (knockout) are not yet available. However, the Brown-Norway-Katholiek (B/N/Ka) rat strain has severe deficiency of both plasma kininogens (12) due to a single point mutation, Ala163 to Thr, which results in defective secretion from the liver. We have shown (7) that occlusive thrombosis resulting from deendothelization of the rat aorta is sixfold more rapid in B/N/Ka rats with kininogen deficiency than in Kitasato rats (B/N/Ki) with normal kininogen, indicating that kininogen is antithrombotic in vivo. However, two limitations of that study should be acknowledged. First, genetic variation may have occurred since the normal rats were not littermate controls. This problem has been solved by backcrossing the kininogen-deficient rats into the Lewis genetic background for six generations (21). Second, no reconstitution was performed with HKa or its domains. Therefore, we have performed a new study in vivo.

We now show that rapid arterial thrombosis characterized by platelet aggregation and fibrin formation in HKd rats can be prevented by infusion of D3 and reversed by a D3-derived peptide. We expressed kininogen D3 and exon 7C product (Fig. 1B) of D3 in Escherichia coli as glutathione S-transferase (GST) fusion proteins and then tested each polypeptide in vivo for its ability to inhibit thrombin-induced platelet aggregation. We also studied the antithrombotic effect of polypeptides without a fusion partner, i.e., HKa produced by plasma kallikrein proteolysis, and a synthetic exon 7C peptide (E7CP), in the kininogen-deficient Lewis (HKd) rat in vivo. Finally, we show that E7CP results in increased inhibition of thrombin, namely, an increase in thrombin-antithrombin (TAT) complexes.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
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Production of kininogen-deficient and wild-type rats on a Lewis genetic background. We previously produced HK-deficient rats on a Lewis genetic background by backcrossing the Lewis rats for six generations with offspring of B/N/Ka x Lewis rats resulting in two new strains, HKd and wild type (WT) (21). In this study we used HKd (HK = 0.08 ± 0.00 U/ml) and WT (HK = 0.99 ± 0.04 U/ml) rats. Each strain was calculated to contain 98.5% Lewis genetic material, where the percentage = 100[1 – (1/2g)] and g is the number of generations. These rats exhibited neither spontaneous thrombosis nor hemorrhage, similar to observations in a human patient with kininogen deficiency (4). In addition, we determined the other three contact factors, Factor XII (FXII), prekallikrein (PK), and FXI (0.97 ± 0.03, 0.99 ± 0.03 and 0.94 ± 0.02 U/ml, respectively) in WT rats. We also determined FXII, PK, and FXI (1.01 ± 0.01, 0.54 ± 0.004 and 0.99 ± 0.004 U/ml, respectively) in HKd rats (21).

Experimental thrombus model in rats. Male HKd and WT-Lewis rats (250–350 g) provided by R. B. Sartor were used in this study to avoid the prothrombotic effects of estrogens. The protocol was approved by the Temple University Committee on Animal Research, and the procedure was similar to that described previously (7). Rats were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg) and maintained by isoflurane inhalation (0.5–1.5%). The right carotid artery and jugular vein were cannulated with polyethylene tubing (PE50) for blood pressure monitoring (using fluid transducer) and intravenous injection, respectively. Baseline aortic flow was measured by placing a perivascular (2SB) ultrasonic flow probe (Transonic Systems, Ithaca, NY) around the aorta immediately above the iliac bifurcation. The entire infrarenal aorta was brushed and denuded of its endothelium with a bronchial cytology brush (C. R. Bard) (7). We maintained a baseline mean arterial blood pressure (MABP) of 90 ± 5 mmHg after anesthesia. All treatments were administered slowly in a volume of 1 ml/kg over 2 min by an intravenous injection as a bolus 10 min before the initiation of endothelial injury. Infusion of GST or test protein increased the baseline MABP 10%, which returned to the basal level within 30 min. Heart rate was increased <10% briefly. Animals were divided into several groups: HKd rats with endothelial injury received GST, GST-D3 (each 6 µM, n = 5), or GST-E7CP (1.5 µM, n = 5). HKd rats with similar endothelial injury received HKa (660 nM, n = 5), plasma concentration of HK (660 nM), or the synthetic peptide E7CP (100, 10, and 3 µM, n = 5). GST fusion proteins were given at the concentrations previously described (1, 16) for inhibiting thrombin activation of platelets in vitro. One hundred micromolar synthetic peptide E7CP was used previously to block thrombin binding to platelet in vitro (1). We tested it in a range of concentrations to find the minimum concentration required to inhibit thrombosis in vivo. One group of WT rats (n = 5) with endothelial injury served as another control. Aortic blood flow was measured and recorded at 5-min intervals just proximal to the aortic bifurcation. The experiment concluded when aortic blood flow dropped to ≤10% of baseline flow or at a total time limit of 240 min of monitoring. One group of HKd rats was infused with GST-E7CP (3 µM, n = 5) 10 min after the injury, and flow was recorded in the same way. Blood samples were then collected from the descending aorta, and animals were euthanized with an overdose of pentobarbital sodium. Infrarenal portions of aorta were saved for histopathology.

Purification of recombinant proteins. The recombinant proteins were purified by the procedure previously described (16, 24). E. coli was grown overnight, and cells were centrifuged at 3,000 g for 20 min at 4°C. The cells were resuspended in PBS containing the protease inhibitors and were disrupted by sonication. The supernatant, after centrifugation, was passed through a glutathione-Sepharose 4B column (Amersham Biosciences). The recombinant fusion protein was eluted with reduced glutathione in Tris·HCl, pH 8.0. The fractions containing the proteins GST, GST-D3, or GST-E7CP were identified by A280 and by SDS-polyacrylamide gel, followed by Coomassie blue staining. In the case of GST-E7CP, the identity was confirmed by protein sequencing. Limulus lysate assay indicated that all recombinant proteins had <0.01 EU/ml of endotoxin. Peptide E7CP was synthesized by the Protein Chemistry Lab, University of Pennsylvania (Philadelphia, PA) and was a single component on high-pressure liquid chromatography. HKa (endotoxin free) was purchased from Enzyme Research Laboratories (South Bend, IN). On reduced SDS-PAGE HKa migrated as a heavy chain of 64 kDa and a light chain of 45 kDa. GST-D3, GST-E7CP, and GST were 43, 29, and 27 kDa, respectively.

Immunohistochemistry. The thoracic and abdominal aorta were collected and fixed in 10% buffered formalin (Fisher Scientific, Fair Lawn, NJ). The specimens were embedded in paraffin and sectioned. The immunoperoxidase technique was performed (Santa Cruz Biotechnology, Santa Cruz, CA) with a mouse monoclonal antibody directed to platelet GPIX antigen to detect aggregated platelet and anti-GST antibody (Sigma Immunochemicals, St. Louis, MO) to detect GST, GST-D3, and GST-E7CP distribution within the vessels. Counterstaining was performed with Gill's hematoxylin (Fisher Scientific). Pictures were taken at magnifications of x100 and x400.

Assays reflecting thrombin activity. Blood was collected in 3.8% sodium citrate (9:1 vol/vol) and centrifuged at 4,000 g for 5 min, and the supernatant was further centrifuged at 12,000 g for 10 min and stored at –70°C. TAT complex was analyzed with an ELISA kit (Enzygnost TAT micro, Dade Behring), which detects thrombin generation in rat plasma (20).

Statistical analysis. Statistical significance was determined by one-way analysis of variance. An all-pairwise multiple comparison procedure (Student-Newman-Keuls method) was applied to evaluate the percentage of blood flow among the different animal groups and the percent binding of GST-E7CP to rat platelets. P values <0.05 were considered significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
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Evaluation of aortic blood flow. We studied the effects of HKa, D3, and its fragment E7CP in acute arterial thrombosis at the site of aortic injury in HKd rats. Occlusion time was defined as the time recorded from the induction of endothelial injury to the time when the blood flow was recorded at ≤10% of the initial flow rate. In our pilot study it was found that some of the HKd rats with injury and GST pretreatment showed improvement of blood flow gradually after a variable period of 10–30 min of occlusion (≤10% of initial flow), whereas in WT injured rats and HKd injured rats treated with kininogen products, the flow was never decreased down to ≤10% of the initial flow rate. In a high-flow vessel like the abdominal aorta it is conceivable that the thrombus can be unstable because of high pressure and pulsatile flow. Since aortas collected after 5 min of occlusion showed stable thrombus histologically, we decided that this rate of flow had to be observed for at least 5 min to assign it as complete occlusion.

In WT rats after endothelial injury a gradual decrease in blood flow was observed over time, but even at 240 min the flow was maintained at 51.4 ± 6.1%. In contrast, in HKd rats within 5 min of endothelial injury flow was decreased down to 20.1 ± 2.0% of baseline value (Fig. 2). At 30 min the mean flow was at 9.8 ± 1.2% of the baseline value. Because each animal in this group fell below 10% of blood flow, the time was recorded and observations of the animal were terminated. When HKd rats were reconstituted with GST-D3 blood flow was maintained over 50% over the entire interval of 5–240 min after endothelial injury. Similar to the HKd+GST-D3 group, the HKd+GST-E7CP and HKd+HKa groups showed a modest decrease in flow after endothelial injury over time, but it never decreased below 36.5%. Figure 2 presents the mean ± SE percentage of baseline flow at 5, 15, 30, and 240 min after endothelial injury for all six groups.


Figure 2
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  		Fig. 2. Percentage of blood flow at different time intervals in each experimental group. Baseline and serial aortic blood flow (5, 15, 30, 240 min) were recorded. Times after injury to 10% of baseline flow [HK deficient (HKd)+GST] or a total time limit of 240 min in the remaining groups are shown. Each point represents the mean ± SE. Numbers of animals in each group are the same as indicated in Fig. 4.

 
All rats (Fig. 3) in the HKd+GST group experienced rapid thrombotic occlusion in contrast to none in the WT+GST group during the 240-min period of observation (P < 0.001). Despite equivalent vascular injury, mean occlusion time was shorter (28 ± 5.2 min) in the HKd+GST group compared with the HKd+GST-D3 and HKd+E7CP groups (>240 min). Treatment with GST-E7CP and HKa also inhibited the occlusion in HKd animals. Differences between GST-only (control) and GST-D3, GST-E7CP, HKa, or synthetic peptide E7CP pretreatments were highly significant (P < 0.001). There was no significant difference between the treated injured HKd and the WT injured rats that received GST alone (240 min).


Figure 3
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  		Fig. 3. Scatter graph showing time to arterial occlusion following endothelial injury in all rats. The wild-type (WT)+GST group ({triangleup}) did not experience thrombotic occlusion during 240 min of observation. The HKd+GST group ({triangledown}) experienced thrombotic occlusion within 28 ± 5.2 min. GST-D3 ({blacktriangleup}), GST-E7CP ({lozenge}), HKa ({circ}), and E7CP ({blacklozenge}) treatment in the HKd rats prevented thrombotic occlusion after injury. All groups were compared with HKd+GST groups (***P = 0.0002). Means (bullet) and SE (bars) are shown; n = number of individual rats.

 
In the HKd+GST-E7CP after injury group of rats, at 5, 15, 30, and 240 min of endothelial injury the mean flow was 21.05 ± 4.4%, 23.1 ± 0.2%, 29.6 ± 0.8%, and 52.50 ± 0.1% of the baseline value, respectively. GST-E7CP infusion 10 min after injury prolonged the flow significantly (P < 0.001) compared with the GST-treated group of HKd rats (Fig. 4). Blood flow within 30 min of injury was higher in the HKd+GST-E7CP before injury group than the flow in the HKd+GST-E7CP after injury group. However, at 240 min there was no significant difference between these two groups.


Figure 4
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  		Fig. 4. Blood flow recording of a representative rat from each experimental group. HKd rats were treated with GST-E7CP 10 min before and after endothelial injury. Data were compared with the GST-pretreated (control) group. Baseline and serial aortic blood flow (5, 15, 30, 240 min) were recorded. HKd+GST rat reached 10% of baseline flow at 30 min, and observations were terminated.

 
Histopathological and immunohistochemistry assessment. The HKd+injury+GST group macroscopically showed laminated thrombi filling the injured arterial segment at the time of retrieval. Histology sections showed 85–90% occlusion of arterial lumina by thrombi containing mixed platelet, fibrin, and entrapped cellular elements. The thrombi were attached to >75% of the vessel wall inner surface (Fig. 5A). There was arterial wall injury involving the endothelium and internal elastic lamina with minimal involvement of the inner muscular layer of the media (the injury involved the whole thickness of the intima). Smooth muscle cell morphology within the media appeared normal. There was no hemorrhage or inflammatory cell infiltrates within the media and adventitial layers of the aortic wall. Moderate to intense antiplatelet immunopositivity within the thrombi and aortic muscular layer was noted.


Figure 5
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  		Fig. 5. Immunohistochemistry detection of platelets and GST-fusion proteins. A: platelet detection by anti-glycoprotein (GP)IX staining. B: detection of GST and GST fusion proteins by anti-GST staining. a, aortic muscular wall; b, aortic lumen; c, endothelial cell lining; d, area of aortic injury; e, platelet adhesion to aortic injury detected with anti-GP-IX-antibody (brown staining); f, thrombus; g, GST distribution detected with anti-GST-antibody (brown staining). Magnification: x100 (left), x400 (right).

 
WT+no injury+GST, WT+injury+GST, HKd+injury+GST-D3, HKd+injury+GST-E7CP, HKd+injury+HKa, and HKd+injury+E7CP groups grossly showed homogeneous dark red blood clots filling the aortic segments at the time of retrieval. Histology sections showed patent arterial lumina with stasis clot (red and white blood cells within a fibrin mesh) completely detached from the arterial wall. Histology sections from the WT+no injury+GST group showed intact endothelial layerand aortic wall. Microscopic evaluation of sections from WT+injury+GST, HKd+injury+GST-D3, HKd+injury+GST-E7CP, HKd+injury+HKa, and HKd+injury+E7CP groups showed arterial wall injury similar to that in the HKd+injury+GST group. All the above groups showed mild to moderate immunopositivity to the antiplatelet antibody within the arterial wall.

GST-E7CP was given after injury, and the flow was monitored for 240 min. The results were identical to GST-E7CP given before injury, suggesting the absence of occlusive thrombus.

Assessment of GST distribution. No GST deposition was detected within the specimen in injured HKd rats treated with GST alone. Mild to moderate anti-GST immunopositivity within the arterial luminal surface and aortic wall was detected in injured HKd rats treated with GST-D3. In HKd injured rats with GST-E7CP pretreatment, mild anti-GST immunopositivity was noted on the arterial luminal surface and within the aortic wall (Fig. 5B).

Evaluation of plasma TAT complexes. Blood samples from HKd+GST+no injury-, HKd+GST+injury-, HKd+injury+GST-E7CP-, HKd+injury+HKa-, and HKd+injury+E7CP-treated groups of rats were taken at the termination of each experiment from the abdominal aorta just proximal to the injured segment. Because GST-D3 and GST-E7CP were equally potent in inhibiting thrombosis, we selected GST-E7CP for these studies. Plasma TAT levels were measured to evaluate systemic activation of coagulation in response to endothelial injury. TAT concentrations were significantly elevated in HKd+GST rats after injury (P < 0.05) (Fig. 6). In HKd rats with endothelial injury, HKa, E7CP, or GST-E7CP pretreatment inhibited thrombin generation as indicated by significant decreases (P < 0.005) in TAT concentrations compared with the GST controls.


Figure 6
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  		Fig. 6. Plasma levels of thrombin-antithrombin (TAT) complexes. Arterial blood samples were collected from HKd+GST, HKd+injury+GST, HKd+injury+GST-E7CP, HKd+injury+HKa, and HKd+injury+E7CP groups of rats immediately after completion of study. TAT concentrations were determined by ELISA. Data are means ± SE; n = 5 in each group, *P < 0.05, **P < 0.005.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We previously showed (19) that HK inhibits thrombin-induced platelet shape change and an increase in intracellular Ca2+ by inhibiting thrombin binding to platelets in vitro. This effect was specific for thrombin since platelets in kininogen-deficient human plasma were not different in their response to either ADP or collagen compared with normal platelet-rich plasma. The action of both HK and LK to inhibit thrombin binding to platelets implies that the common heavy chain is involved. D3 inhibited 125I-labeled HK binding to platelets, indicating that this domain contains a cell-binding region (14). Recombinant D3 inhibited thrombin-induced aggregation of platelets with only twofold less affinity than purified HK (16), confirming that a major site for inhibition resides in D3. More specifically, the recombinant polypeptide coded by the COOH-terminal portion of exon 7, K270-Q292, exhibited in vitro potency equivalent to HK or D3. The synthetic peptide K270-Q292 inhibited thrombin-induced platelet activation at low thrombin concentrations by inhibiting the binding of thrombin to the GPIb-IX-V complex on platelets. Antibodies to and ligands of GPIb inhibited HK binding to platelets. Finally, HK inhibited binding of thrombin to fibroblasts transfected with GPIb-IX-V. These findings suggest that HK, LK, D3, and its peptides may block thrombin binding to its high-affinity site on GPIb (1). Thus kininogens contribute to the physiological state where no platelet activation is evident. They exert their influence without the requirement for cleavage since HK and HKa both exhibit this property. Because thrombin binding to GPIb enhances its cleavage of protease-activated receptor (PAR)-1 on intact platelets (8), interference with this binding would be expected to inhibit platelet activation.

The localized alteration in our selective endothelial denudation model (7) closely imitates the vascular injury experienced in patients after angioplasty. Platelet activation, thrombin production, and fibrin formation were documented in this animal model of aortic thrombus formation. The B/N/Ka rat strain has a severe deficiency of LK and HK and showed rapid thrombotic occlusion compared with WT animals (7). To ensure an identical genetic background in the present study we used WT and HKd rats backcrossed to Lewis for six generations, giving a 98.5% Lewis genetic background in all animals. We observed rapid onset of aortic thrombosis (30 min) with GST-treated HKd animals with limited arterial injury, whereas WT rats in the Lewis genetic background showed lack of thrombotic occlusion even at 240 min, as previously described (7).

GST-D3 or GST-E7CP pretreatment in HKd rats inhibited thrombosis significantly compared with HKd GST-treated rats. Our findings indicate that the kininogen D3 plays an important role in preventing induction of thrombosis in WT rats. When GST-E7CP was infused 10 min after endothelial injury it still inhibited the formation of an occlusive thrombus. The blood flow was maintained at 240 min of observation. Within the first 30 min of endothelial injury the flow was significantly decreased because the GST-E7CP administration had been delayed. This finding indicates that kininogen derivatives inhibit the formation of thrombus in addition to preventing the induction of thrombus formation. Profound antithrombotic effects of both GST-D3 and GST-E7CP were found in kininogen-deficient rats with endothelial injury, indicating that the amino acid sequence K270-Q292 derived from D3 is equally potent to HK D3. For GST and GST-D3 the maximum calculated plasma concentration immediately after infusion was ~6 µM, and for GST-E7CP it was 1.5 µM. Thus the concentrations of GST and GST-D3 were approximately the same, indicating that GST itself had no effect and that the polypeptide D3 was responsible for the inhibition of arterial thrombosis. GST-E7CP was an equally potent antithrombotic agent as judged by its ability to maintain flow but was effective at one-fourth the concentration of GST-D3. Lower concentrations of GST-D3 were not tested, so we can conclude that GST-E7CP is at least as potent on a molar basis as the larger parent molecule. Thus this 23-amino acid polypeptide contains virtually all the antithrombotic activity of HK-D3. To further test this conclusion, we used HKa and E7CP, neither of which contained GST. HKa at a concentration of 660 nM was found to be protective against thrombosis, whereas the synthetic peptide E7CP was a potent antithrombotic peptide at concentrations of 100 and 10 µM, but E7CP failed to inhibit thrombosis at a concentration of 3 µM. We speculate that the higher concentration of E7CP than GST-E7CP needed in vivo resulted from a short half-life of the former due to its low molecular weight and probable rapid renal clearance. When GST-E7CP was infused after injury was induced, twice the concentration was needed to maintain the flow compared with the concentration used before the injury.

The GST fusion proteins with D3 and derivatives of D3 were bound to the injured vessel, but GST itself did not bind (Fig. 5B). The fusion protein GST-E7CP binds to platelets, indicating that E7CP is the critical sequence. These observations indicate that only the kininogen moiety, but not GST, binds to endothelial cells.

The development of thrombus formation in this model involves platelets, coagulation factors, fibrinolytic proteases, and the vessel wall after deendothelialization. Platelet activation, specifically aggregation after endothelial injury, is an important component of the occlusive thrombus in vivo. Thrombin is a strong mediator of platelet activation. we showed previously (16) in vitro that the inhibitory site is present in D3, confined to the COOH-terminal portion of the peptide encoded by the 3' sequence of exon 7. The present study shows that the same sequence is antithrombotic in vivo. By staining platelets with anti-human GPIX, which is specific for platelets, we have shown that the thrombi contain large numbers of aggregated platelets after endothelial injury in kininogen-deficient rats. In contrast, normal rats showed only a rim of platelets attached to the media, indicating platelet adhesion (presumably mediated by von Willebrand factor) without platelet aggregation, consistent with the in vitro ability of kininogens to inhibit platelet aggregation.

Contrary to its effect on normal platelets, HK (2 µmol/l) did not inhibit the thrombin-induced aggregation of Bernard-Soulier platelets, which lack the GPIb-IX-V complex, suggesting that kininogen interacts either directly or indirectly with that complex (1). Previous studies have shown that GPIb is the high-affinity binding site for thrombin with a maximum density of binding sites of 50 sites/platelet and a Kd of 0.3 nmol/l (10, 13). Our results using GST-D3 and E7CP in vivo are consistent with the in vitro findings and support the concept that kininogen D3 is antithrombotic and the E7CP region of D3 is able to inhibit the subsequent platelet aggregation.

Our focus on D3 in this study does not rule out participation of other kininogen domains in the in vivo model we have used. D4 fragments, including bradykinin (BK)1–5, were shown to be inhibitors of thrombin-induced platelet aggregation by preventing thrombin from cleaving its cloned receptor (PAR-1) (11). In our study we used HKa and the E7CP peptide lacking bradykinin. Other domains of HK also exhibit antiadhesive and potentially antithrombotic properties, especially in the two-chain, kinin-free form (HKa) in vitro. Considerable quantities of HK/HKa are found to be associated with the surface of platelets or within platelet-released products (22, 23). HKa, and especially D5, blocks platelet adhesion to vitronectin bound to the surface of fibrin fibrils (18). Moreover, D5 and particularly the COOH-terminal region Gly486-Lys502 were identified as potent inhibitors (3) of platelet aggregation by blocking the {alpha}IIbbeta3-vitronectin interaction. In addition, the light chain (D5–D6) of HKa can enhance endothelial cell-mediated fibrinolysis (17). D5 serves as the binding site on HKa to endothelial cells, and D6 has the binding site for PK. Thus multiple domains may mediate the antithrombotic action of HK and HKa.

Endothelial denudation causes exposure of collagen and of tissue factor, which is constitutively expressed on vascular smooth muscle. These stimuli result in platelet adhesion followed by thrombin-induced platelet aggregation and degranulation. In WT rats the presence of HK and LK prevents thrombin from binding to GPIb receptors, thereby modulating platelet activation and inhibiting thrombin generation. In HKd rats this important negative regulation does not occur, and thus we observed a significant increase in plasma TAT in rats with kininogen deficiency after injury. When kininogen-deficient rats were pretreated with GST-D3 and GST-E7CP before endothelial injury, significant decrease in TAT levels was demonstrated. Presumably since activated platelets are a major locus for thrombin formation the decrease in thrombin binding releases the feedback of thrombin by activating platelets to form more thrombin. Similarly, treatment of the HKd rats with HKa or E7CP also decreased TAT compared with the GST group. These results suggest that D3 and E7CP inhibited thrombosis in HKd rats.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Institutes of Health Grants R01-CA-83121-08 and T32-HL-00777-13.


   ACKNOWLEDGMENTS
 
We thank Dr. Bo Liu for skillful maintenance of the rat colony. We also thank Princess Graham for help in the manuscript and graphics preparation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. W. Colman, Sol Sherry Thrombosis Research Center, Temple Univ. School of Medicine, 3400 N. Broad St., Rm. 418 OMS, Philadelphia, PA 19140 (e-mail: colmanr{at}temple.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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